Saturation mutagenesis in directed evolution

ABSTRACT

Disclosed is a rapid and facilitated method of producing from a parentlal template polynucleotide, a set of mutagenized progeny polynculeotides whereby at each original codon position there is produced at least one substitute codon encoding each of the 20 naturally encoded amino acids. Accordingly, there is also provided a method of producing from a parental template polypeptide, a set of mutagenized progeny polypeptides wherein each of the 20 naturally encoded amino acids is represented at each original amino acid position. The method provided is termed site-saturation mutagenesis, or simply saturation mutagenesis, and can be used in combination with other mutagenization processes, such as, for example, a process wherein two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment. Also provided are vector and expression vehicles incuding such polynucleotides, polypeptides expressed by the hybrid polynucleotides and a method for screening for hybrid polypeptides.

FIELD OF THE INVENTION

This invention relates to the field of protein engineering. Morespecifically, this relates to a directed evolution method for preparinga polynucleotides encoding polypeptide, which method comprises the stepof generating site-directed mutagenesis optionally in combination withthe step of polynucleotide chimerization, the step of selecting forpotentially desirable progeny molecules (which may then be screenedfurther), and the step of screening the polynucleotides for theproduction of polypeptide(s) having a useful property.

In a particular aspect, the present invention is relevant to enzymes,particularly to thermostable enzymes, and to their generation bydirected evolution. More particularly, the present invention relates tothermostable enzymes which are stable at high temperature and which haveimproved activity at lower temperatures.

BACKGROUND

Harvesting the full potential of nature's diversity can include both thestep of discovery and the step of optimizing what is discovered. Forexample, the step of discovery allows one to mine biological moleculesthat have industrial utility. However, for certain industrial needs, itis advantageous to further modify these enzymes experimentally toachieve properties beyond what natural evolution has provided and islikely to provide in the near future.

The process, termed directed evolution, of experimentally modifying abiological molecule towards a desirable property, can be achieved bymutagenizing one or more parental molecular templates and idendifyingany desirable molecules among the progeny molecules. However, currentlyavailable technologies used in directed evolution have severalshortfalls. Among these shortfalls are:

-   -   1) Site-directed mutagenesis technologies, such as sloppy or        low-fidelity PCR, are ineffective for systematically achieving        at each position (site) along a polypeptide sequence the full        (saturated) range of possible mutations (i.e. all possible amino        acid substitutions).    -   2) There is no relatively easy systematic means for rapidly        analyzing the large amount of information that can be contained        in a molecular sequence and in the potentially colossal number        or progeny molecules that could be conceivably obtained by the        directed evolution of one or more molecular templates.    -   3) There is no relatively easy systematic means for providing        comprehensive empirical information relating structure to        function for molecular positions.    -   4) There is no easy systematic means for incorporating internal        controls in certain mutagenesis (e.g. chimerization) procedures.    -   5) There is no easy systematic means to select for specific        progeny molecules, such as full-length chimeras, from among        smaller partial sequences.

Molecular mutagenesis occurs in nature and has resulted in thegeneration of a wealth of biological compounds that have shown utilityin certain industrial applications. However, evolution in nature oftenselects for molecular properties that are discordant with many unmetindustrial needs. Additionally, it is often the case that when anindustrially useful mutations would otherwise be favored at themolecular level, natural evolution often overrides the positiveselection of such mutations when there is a concurrent detriment to anorganism as a whole (such as when a favorable mutation is accompanied bya detrimental mutation). Additionally still, natural evolution is slow,and places high emphasis on fidelity in replication. Finally, naturalevolution prefers a path paved mainly by beneficial mutations whiletending to avoid a plurality of successive negative mutations, eventhough such negative mutations may prove beneficial when combined, ormay lead—through a circuitous route—to final state that is beneficial.

Directed evolution, on the other hand, can be performed much morerapidly and aimed directly at evolving a molecular property that isindustrially desirable where nature does not provide one.

An exceedingly large number of possibilities exist for purposeful andrandom combinations of amino acids within a protein to produce usefulhybrid proteins and their corresponding biological molecules encodingfor these hybrid proteins, i.e., DNA, RNA. Accordingly, there is a needto produce and screen a wide variety of such hybrid proteins for adesirable utility, particularly widely varying random proteins.

The complexity of an active sequence of a biological macromolecule(e.g., polynucleotides, polypeptides, and molecules that are comprisedof both polynucleotide and polypeptide sequences) has been called itsinformation content (“IC”), which has been defined as the resistance ofthe active protein to amino acid sequence variation (calculated from theminimum number of invariable amino acids (bits) required to describe afamily of related sequences with the same function). Proteins that aremore sensitive to random mutagenesis have a high information content.

Molecular biology developments, such as molecular libraries, haveallowed the identification of quite a large number of variable bases,and even provide ways to select functional sequences from randomlibraries. In such libraries, most residues can be varied (althoughtypically not all at the same time) depending on compensating changes inthe context. Thus, while a 100 amino acid protein can contain only 2,000different mutations, 20¹⁰⁰ sequence combinations are possible.

Information density is the IC per unit length of a sequence. Activesites of enzymes tend to have a high information density. By contrast,flexible linkers of information in enzymes have a low informationdensity.

Current methods in widespread use for creating alternative proteins in alibrary format are error-prone polymerase chain reactions and cassettemutagenesis, in which the specific region to be optimized is replacedwith a synthetically mutagenized oligonucleotide. In both cases, asubstantial number of mutant sites are generated around certain sites inthe original sequence.

Error-prone PCR uses low-fidelity polymerization conditions to introducea low level of point mutations randomly over a long sequence. In amixture of fragments of unknown sequence, error-prone PCR can be used tomutagenize the mixture. The published error-prone PCR protocols sufferfrom a low processivity of the polymerase. Therefore, the protocol isunable to result in the random mutagenesis of an average-sized gene.This inability limits the practical application of error-prone PCR. Somecomputer simulations have suggested that point mutagenesis alone mayoften be too gradual to allow the large-scale block changes that arerequired for continued and dramatic sequence evolution. Further, thepublished error-prone PCR protocols do not allow for amplification ofDNA fragments greater than 0.5 to 1.0 kb, limiting their practicalapplication. In addition, repeated cycles of error-prone PCR can lead toan accumulation of neutral mutations with undesired results, such asaffecting a protein's immunogenicity but not its binding affinity.

In oligonucleotide-directed mutagenesis, a short sequence is replacedwith a synthetically mutagenized oligonucleotide. This approach does notgenerate combinations of distant mutations and is thus notcombinatorial. The limited library size relative to the vast sequencelength means that many rounds of selection are unavoidable for proteinoptimization. Mutagenesis with synthetic oligonucleotides requiressequencing of individual clones after each selection round followed bygrouping them into families, arbitrarily choosing a single family, andreducing it to a consensus motif. Such motif is resynthesized andreinserted into a single gene followed by additional selection. Thisstep process constitutes a statistical bottleneck, is labor intensive,and is not practical for many rounds of mutagenesis.

Error-prone PCR and oligonucleotide-directed mutagenesis are thus usefulfor single cycles of sequence fine tuning, but rapidly become toolimiting when they are applied for multiple cycles.

Another limitation of error-prone PCR is that the rate of down-mutationsgrows with the information content of the sequence. As the informationcontent, library size, and mutagenesis rate increase, the balance ofdown-mutations to up-mutations will statistically prevent the selectionof further improvements (statistical ceiling).

In cassette mutagenesis, a sequence block of a single template istypically replaced by a (partially) randomized sequence. Therefore, themaximum information content that can be obtained is statisticallylimited by the number of random sequences (i.e., library size). Thiseliminates other sequence families which are not currently best, butwhich may have greater long term potential.

Also, mutagenesis with synthetic oligonucleotides requires sequencing ofindividual clones after each selection round. Thus, such an approach istedious and impractical for many rounds of mutagenesis.

Thus, error-prone PCR and cassette mutagenesis are best suited, and havebeen widely used, for fine-tuning areas of comparatively low informationcontent. One apparent exception is the selection of an RNA ligaseribozyme from a random library using many rounds of amplification byerror-prone PCR and selection.

In nature, the evolution of most organisms occurs by natural selectionand sexual reproduction. Sexual reproduction ensures mixing andcombining of the genes in the offspring of the selected individuals.During meiosis, homologous chromosomes from the parents line up with oneanother and cross-over part way along their length, thus randomlyswapping genetic material. Such swapping or shuffling of the DNA allowsorganisms to evolve more rapidly.

In recombination, because the inserted sequences were of proven utilityin a homologous environment, the inserted sequences are likely to stillhave substantial information content once they are inserted into the newsequence.

The term Applied Molecular Evolution (“AME”) means the application of anevolutionary design algorithm to a specific, useful goal. While manydifferent library formats for AME have been reported forpolynucleotides, peptides and proteins (phage, lad and polysomes), noneof these formats have provided for recombination by random cross-oversto deliberately create a combinatorial library.

Theoretically there are 2,000 different single mutants of a 100 aminoacid protein. However, a protein of 100 amino acids has 20¹⁰⁰ possiblesequence combinations, a number which is too large to exhaustivelyexplore by conventional methods. It would be advantageous to develop asystem which would allow generation and screening of all of thesepossible combination mutations.

Some workers in the art have utilized an in vivo site specificrecombination system to generate hybrids of combine light chain antibodygenes with heavy chain antibody genes for expression in a phage system.However, their system relies on specific sites of recombination and islimited accordingly. Simultaneous mutagenesis of antibody CDR regions insingle chain antibodies (scFv) by overlapping extension and PCR havebeen reported.

Others have described a method for generating a large population ofmultiple hybrids using random in vivo recombination. This methodrequires the recombination of two different libraries of plasmids, eachlibrary having a different selectable marker. The method is limited to afinite number of recombinations equal to the number of selectablemarkers existing, and produces a concomitant linear increase in thenumber of marker genes linked to the selected sequence(s).

In vivo recombination between two homologous, but truncated,insect-toxin genes on a plasmid has been reported as a method ofproducing a hybrid gene. The in vivo recombination of substantiallymismatched DNA sequences in a host cell having defective mismatch repairenzymes, resulting in hybrid molecule formation has been reported.

SUMMARY OF THE INVENTION

This invention relates generally to the field of nucleic acidengineering and correspondingly encoded recombinant protein engineering.More particularly, the invention relates to the directed evolution ofnucleic acids and screening of clones containing the evolved nucleicacids for resultant activity(ies) of interest, such nucleic acidactivity(ies) &/or specified protein, particularly enzyme, activity(ies)of interest.

This invention relates generally to a method of: 1) preparing a progenygeneration molecule (including a molecule that is comprised of apolynucleotide sequence, a molecules that is comprised of a polypeptidesequence, and a molecules that is comprised in part of a polynucleotidesequence and in part of a polypeptide sequence), that is mutagenized toachieve at least one point mutation, addition, deletion, &/orchimerization, from one or more ancestral or parental generationtemplate(s); 2) screening the progeny generation molecule—preferablyusing a high throughput method—for at least one property of interest(such as an improvement in an enzyme activity or an increase instability or a novel chemotherapeutic effect); 3) optionally obtaining&/or cataloguing structural &/or and functional information regardingthe parental &/or progeny generation molecules; and 4) optionallyrepeating any of steps 1) to 3).

In a preferred embodiment, there is generated (e.g. from a parentpolynucleotide template)—in what is termed “codon site-saturationmutagenesis”—a progeny generation of polynucleotides, each having atleast one set of up to three contiguous point mutations (i.e. differentbases comprising a new codon), such that every codon (or every family ofdegenerate codons encoding the same amino acid) is represented at eachcodon position. Corresponding to—and encoded by—this progeny generationof polynucleotides, there is also generated a set of progenypolypeptides, each having at least one single amino acid point mutation.In a preferred aspect, there is generated—in what is termed “amino acidsite-saturation mutagenesis”—one such mutant polypeptide for each of the19 naturally encoded polypeptide-forming alpha-amino acid substitutionsat each and every amino acid position along the polypeptide. Thisyields—for each and every amino acid position along the parentalpolypeptide—a total of 20 distinct progeny polypeptides including theoriginal amino acid, or potentially more than 21 distinct progenypolypeptides if additional amino acids are used either instead of or inaddition to the 20 naturally encoded amino acids

Thus, in another aspect, this approach is also serviceable forgenerating mutants containing—in addition to &/or in combination withthe 20 naturally encoded polypeptide-forming alpha-amino acids—otherrare &/or not naturally-encoded amino acids and amino acid derivatives.In yet another aspect, this approach is also serviceable for generatingmutants by the use of—in addition to &/or in combination with natural orunaltered codon recognition systems of suitable hosts—altered,mutagenized, &/or designer codon recognition systems (such as in a hostcell with one or more altered tRNA molecules).

In yet another aspect, this invention relates to recombination and morespecifically to a method for preparing polynucleotides encoding apolypeptide by a method of in vivo reassortment of polynucleotidesequences containing regions of partial homology, assembling thepolynucleotides to form at least one polynucleotide and screening thepolynucleotides for the production of polypeptide(s) having a usefulproperty.

In yet another preferred embodiment, this invention is serviceable foranalyzing and cataloguing—with respect to any molecular property (e.g.an enzymatic activity) or combination of properties allowed by currenttechnology—the effects of any mutational change achieved (includingparticularly saturation mutagenesis). Thus, a comprehensive method isprovided for determining the effect of changing each amino acid in aparental polypeptide into each of at least 19 possible substitutions.This allows each amino acid in a parental polypeptide to becharacterized and catalogued according to its spectrum of potentialeffects on a measurable property of the polypeptide.

In another aspect, the method of the present invention utilizes thenatural property of cells to recombine molecules and/or to mediatereductive processes that reduce the complexity of sequences and extentof repeated or consecutive sequences possessing regions of homology.

It is an object of the present invention to provide a method forgenerating hybrid polynucleotides encoding biologically active hybridpolypeptides with enhanced activities. In accomplishing these and otherobjects, there has been provided, in accordance with one aspect of theinvention, a method for introducing polynucleotides into a suitable hostcell and growing the host cell under conditions that produce a hybridpolynucleotide.

In another aspect of the invention, the invention provides a method forscreening for biologically active hybrid polypeptides encoded by hybridpolynucleotides. The present method allows for the identification ofbiologically active hybrid polypeptides with enhanced biologicalactivities.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the variety of likely mutations inpolypeptide that result from the introduction of single point mutationsin a polynucleotide encoding said polypeptide by a method such aserror-prone PCR. Because replicative errors in a polynucleotidesequence, such as those introduced using error-prone PCR are unlikely togenerate two—much less three—contiguous nucleotide changes, said methodsare unlikely to achieve more than 5-7 (on average) codon changes at eachcodon position. Illustrated is the poor ability of this approach forachieving all possible amino acid changes at each amino acid site alongthe polypeptide. In contrast, the site-saturation mutagenesis approachprovided by the instant invention does achieve a range of codonsubstitutions (preferably comprising the 32 codons represented by thedegenerate cassette sequence N,N,G/T) so as to achieve all possibleamino acid changes at each amino acid site along a polypeptide.

FIG. 2 is a diagram illustrating the use of a site-saturationmutagenesis approach for achieving all possible amino acid changes ateach amino acid site along the polypeptide. The oligos used arecomprised of a homologous sequence, a triplet sequence composed ofdegenerate N,N, G/T, and another homologous sequence. Thus, thedegeneracy of each oligo is derived from the degeneracy of the N,N, G/Tcassette contained therein. The resultant polymerization products fromthe use of such oligos include all possible amino acid changes at eachamino acid site along the polypeptide, because the N,N, G/T sequence isable to code for all 20 amino acids. As shown, a separate degenerateoligo is used for mutagenizing each codon in a polynucleotide encoding apolypeptide.

DEFINITIONS OF TERMS

In order to facilitate understanding of the examples provided herein,certain frequently occurring methods and/or terms will be described.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds; an array of spatially localized compounds (e.g.,a VLSIPS peptide array, polynucleotide array, and/or combinatorial smallmolecule array), biological macromolecule, a bacteriophage peptidedisplay library, a bacteriophage antibody (e.g., scFv) display library,a polysome peptide display library, or an extract made form biologicalmaterials such as bacteria, plants, fungi, or animal (particularmammalian) cells or tissues. Agents are evaluated for potential activityas anti-neoplastics, anti-inflammatories or apoptosis modulators byinclusion in screening assays described hereinbelow. Agents areevaluated for potential activity as specific protein interactioninhibitors (i.e., an agent which selectively inhibits a bindinginteraction between two predetermined polypeptides but which doe snotsubstantially interfere with cell viability) by inclusion in screeningassays described hereinbelow.

The term “amino acid” as used herein refers to any organic compound thatcontains an amino group (—NH₂) and a carboxyl group (—COOH); preferablyeither as free groups or alternatively after condensation as part ofpeptide bonds. The “twenty naturally encoded polypeptide-formingalpha-amino acids” are understood in the art and refer to: alanine (alaor A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp orD), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gln orQ), glycine (gly or G), histidine (his or H), isoleucine (ile or I),leucine (leu or L), lysine (lys or K), methionine (met or M),phenylalanine (phe or F), proline (pro or P), serine (ser or S),threonine (thr or T), tryptophan (trp or W), tyrosine (tyr or Y), andvaline (val or V).

The term “amplification” means that the number of copies of apolynucleotide is increased.

The term “antibody”, as used herein, refers to intact immunoglobulinmolecules, as well as fragments of immunoglobulin molecules, such asFab, Fab′, (Fab′)₂, Fv, and SCA fragments, that are capable of bindingto an epitope of an antigen. These antibody fragments, which retain someability to selectively bind to an antigen (e.g., a polypeptide antigen)of the antibody from which they are derived, can be made using wellknown methods in the art (see, e.g., Harlow and Lane, supra), and aredescribed further, as follows.

-   -   (1) An Fab fragment consists of a monovalent antigen-binding        fragment of an antibody molecule, and can be produced by        digestion of a whole antibody molecule with the enzyme papain,        to yield a fragment consisting of an intact light chain and a        portion of a heavy chain.    -   (2) An Fab′ fragment of an antibody molecule can be obtained by        treating a whole antibody molecule with pepsin, followed by        reduction, to yield a molecule consisting of an intact light        chain and a portion of a heavy chain. Two Fab′ fragments are        obtained per antibody molecule treated in this manner.    -   (3) An (Fab′)₂ fragment of an antibody can be obtained by        treating a whole antibody molecule with the enzyme pepsin,        without subsequent reduction. A (Fab′)₂ fragment is a dimer of        two Fab′ fragments, held together by two disulfide bonds.    -   (4) An Fv fragment is defined as a genetically engineered        fragment containing the variable region of a light chain and the        variable region of a heavy chain expressed as two chains.    -   (5) An single chain antibody (“SCA”) is a genetically engineered        single chain molecule containing the variable region of a light        chain and the variable region of a heavy chain, linked by a        suitable, flexible polypeptide linker.

A molecule that has a “chimeric property” is a molecule that is: 1) inpart homologous and in part heterologous to a first reference molecule;while 2) at the same time being in part homologous and in partheterologous to a second reference molecule; without 3) precluding thepossibility of being at the same time in part homologous and in partheterologous to still one or more additional reference molecules. In anon-limiting embodiment, a chimeric molecule may be prepared byassemblying a reassortment of partial molecular sequences. In anon-limiting aspect, a chimeric polynucleotide molecule may be preparedby synthesizing the chimeric polynucleotide using plurality of moleculartemplates, such that the resultant chimeric polynucleotide hasproperties of a plurality of templates.

The term “cognate” as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example,but not limitation, in the human genome the human CD4 gene is thecognate gene to the mouse 3d4 gene, since the sequences and structuresof these two genes indicate that they are highly homologous and bothgenes encode a protein which functions in signaling T cell activationthrough MHC class II-restricted antigen recognition.

A “comparison window,” as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482 by the homologyalignment algorithm of Needlemen and Wuncsch J. Mol. Biol. 48: 443(1970), by the search of similarity method of Pearson and Lipman Proc.Natl. Acad. Sci. (U.S.A 85: 2444 (1988), by computerized implementationsof these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package Release 7.0, Genetics Computer Group. 575Science Dr., Madison, Wis.), or by inspection, and the best alignment(i.e., resulting in the highest percentage of homology over thecomparison window) generated by the various methods is selected.

As used herein, the term “complementarity-determining region” and “CDR”refer to the art-recognized term as exemplified by the Kabat and ChothiaCDR definitions also generally known as supervariable regions orhypervariable loops (Chothia and Leks, 1987; Clothia et al., 1989; Kabatet al., 1987; and Tramontano et al., 1990). Variable region domainstypically comprise the amino-terminal approximately 105-115 amino acidsof a naturally-occurring immunoglobulin chain (e.g., amino acids 1-110),although variable domains somewhat shorter or longer are also suitablefor forming single-chain antibodies.

“Conservative amino acid substitutions” refer to the interchangeabilityof residues having similar side chains. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference “TATAC” and iscomplementary to a reference sequence “GTATA.”

The term “degrading effective” amount refers to the amount of enzymewhich is required to process at least 50% of the substrate, as comparedto substrate not contacted with the enzyme. Preferably, at least 80% ofthe substrate is degraded.

As used herein, the term “defined sequence framework” refers to a set ofdefined sequences that are selected on a non-random basis, generally onthe basis of experimental data or structural data; for example, adefined sequence framework may comprise a set of amino acid sequencesthat are predicted to form a B-sheet structure or may comprise a leucinezipper heptad repeat motif, a zinc-finger domain, among othervariations. A “defined sequence kernal” is a set of sequences whichencompass a limited scope of variability. Whereas (1) a completelyrandom 10-mer sequence of the 20 conventional amino acids can be any of(20)¹⁰ sequences, and (2) a pseudorandom 10-mer sequence of the 20conventional amino acids can be any of (20)¹⁰ sequences but will exhibita bias for certain residues at certain positions and/or overall, (3) adefined sequence kernal is a subset of sequences if each residueposition was allowed to be any of the allowable 20 conventional aminoacids (and/or allowable unconventional amino/imino acids). A definedsequence kernal generally comprises variant and invariant residuepositions and/or comprises variant residue positions which can comprisea residue selected from a defined subset of amino acid residues), andthe like, either segmentally or over the entire length of the individualselected library member sequence. Defined sequence kernels can refer toeither amino acid sequences or polynucleotide sequences. Of illustrationand not limitation, the sequences (NNK)₁₀ and (NNM)₁₀, wherein Nrepresents A, T, G, or C; K represents G or T; and M represents A or C,are defined sequence kernels.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a gel to isolate the desired fragment.

The term “DNA shuffling” is used herein to indicate recombinationbetween substantially homologous but non-identical sequences, in someembodiments DNA shuffling may involve crossover via non-homologousrecombination, such as via cer/lox and/or flp/frt systems and the like.

As used in this invention, the term “epitope” refers to an antigenicdeterminant on an antigen, such as a phytase polypeptide, to which theparatope of an antibody, such as an phytase-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics. As used herein “epitope” refers tothat portion of an antigen or other macromolecule capable of forming abinding interaction that interacts with the variable region binding bodyof an antibody. Typically, such binding interaction is manifested as anintermolecular contact with one or more amino acid residues of a CDR.

The terms “fragment”, “derivative” and “analog” when referring to areference polypeptide comprise a polypeptide which retains at least onebiological function or activity that is at least essentially same asthat of the reference polypeptide. Furthermore, the terms “fragment”,“derivative” or “analog” are exemplified by a “pro-form” molecule, suchas a low activity proprotein that can be modified by cleavage to producea mature enzyme with significantly higher activity.

A method is provided herein for producing from a template polypeptide aset of progeny polypeptides in which a “full range of single amino acidsubstitutions” is represented at each amino acid position. As usedherein, “full range of single amino acid substitutions” is in referenceto the naturally encoded 20 naturally encoded polypeptide-formingalpha-amino acids, as described herein.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

“Genetic instability”, as used herein, refers to the natural tendency ofhighly repetitive sequences to be lost through a process of reductiveevents generally involving sequence simplification through the loss ofrepeated sequences. Deletions tend to involve the loss of one copy of arepeat and everything between the repeats.

The term “heterologous” means that one single-stranded nucleic acidsequence is unable to hybridize to another single-stranded nucleic acidsequence or its complement. Thus areas of heterology means that areas ofpolynucleotides or polynucleotides have areas or regions within theirsequence which are unable to hybridize to another nucleic acid orpolynucleotide. Such regions or areas are for example areas ofmutations.

The term “homologous” or “homeologous” means that one single-strandednucleic acid nucleic acid sequence may hybridize to a complementarysingle-stranded nucleic acid sequence. The degree of hybridization maydepend on a number of factors including the amount of identity betweenthe sequences and the hybridization conditions such as temperature andsalt concentrations as discussed later. Preferably the region ofidentity is greater than about 5 bp, more preferably the region ofidentity is greater than 10 bp.

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, alsocalled CDR's. The extent of the framework region and CDR's have beenprecisely defined (see, “Sequences of Proteins of ImmunologicalInterest,” Kabat et al., 1987). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecie. As used herein, a “human framework region” is a framework regionthat is substantially identical (about 85 or more, usually 90-95 ormore) to the framework region of a naturally occurring humanimmunoglobulin. the framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR's. The CDR's are primarilyresponsible for binding to an epitope of an antigen.

The benefits of this invention extend to “industrial applications” (orindustrial processes), which term is used to include applications incommercial industry proper (or simply industry) as well asnon-commercial industrial applications (e.g. biomedical research at anon-profit institution). Relevant applications include those in areas ofdiagnosis, medicine, agriculture, manufacturing, and academia.

The term “identical” or “identity” means that two nucleic acid sequenceshave the same sequence or a complementary sequence. Thus, “areas ofidentity” means that regions or areas of a polynucleotide or the overallpolynucleotide are identical or complementary to areas of anotherpolynucleotide or the polynucleotide.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or enzymepresent in a living animal is not isolated, but the same polynucleotideor enzyme, separated from some or all of the coexisting materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or enzymes could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

By “isolated nucleic acid” is meant a nucleic acid, e.g., a DNA or RNAmolecule, that is not immediately contiguous with the 5′ and 3′ flankingsequences with which it normally is immediately contiguous when presentin the naturally occurring genome of the organism from which it isderived. The term thus describes, for example, a nucleic acid that isincorporated into a vector, such as a plasmid or viral vector; a nucleicacid that is incorporated into the genome of a heterologous cell (or thegenome of a homologous cell, but at a site different from that at whichit naturally occurs); and a nucleic acid that exists as a separatemolecule, e g., a DNA fragment produced by PCR amplification orrestriction enzyme digestion, or an RNA molecule produced by in vitrotranscription. The term also describes a recombinant nucleic acid thatforms part of a hybrid gene encoding additional polypeptide sequencesthat can be used, for example, in the production of a fusion protein.

As used herein “ligand” refers to a molecule, such as a random peptideor variable segment sequence, that is recognized by a particularreceptor. As one of skill in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis et al, 1982, p.146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

As used herein, “linker” or “spacer” refers to a molecule or group ofmolecules that connects two molecules, such as a DNA binding protein anda random peptide, and serves to place-the two molecules in a preferredconfiguration, e.g., so that the random peptide can bind to a receptorwith minimal steric hindrance from the DNA binding protein.

As used herein, a “molecular property to be evolved” includes referenceto molecules comprised of a polynucleotide sequence, molecules comprisedof a polypeptide sequence, and molecules comprised in part of apolynucleotide sequence and in part of a polypeptide sequence.Particularly relevant—but by no means limiting—examples of molecularproperties to be evolved include enzymatic activities at specifiedconditions, such as related to temperature; salinity; pressure; pH; andconcentration of glycerol, DMSO, detergent, &/or any other molecularspecies with which contact is made in a reaction environment. Additionalparticularly relevant—but by no means limiting—examples of molecularproperties to be evolved include stabilities—e.g. the amount of aresidual molecular property that is present after a specified exposuretime to a specified environment, such as may be encountered duringstorage.

The term “mutations” means changes in the sequence of a wild-typenucleic acid sequence or changes in the sequence of a peptide. Suchmutations may be point mutations such as transitions or transversions.The mutations may be deletions, insertions or duplications.

As used herein, the degenerate “N,N,G/T” nucleotide sequence represents32 possible triplets, where “N” can be A, C, G or T.

The term “naturally-occurring” as used herein as applied to the objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring. Generally, the term naturally occurring refers toan object as present in a non-pathological (un-diseased) individual,such as would be typical for the species.

As used herein, a “nucleic acid molecule” is comprised of at least onebase or one base pair, depending on whether it is single-stranded ordouble-stranded, respectively. Furthermore, a nucleic acid molecule maybelong exclusively or chimerically to any group of nucleotide-containingmolecules, as exemplified by, but not limited to, the following groupsof nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomicnucleic acids, naturally occurring and not naturally occurring nucleicacids, and synthetic nucleic acids. This includes, by way ofnon-limiting example, nucleic acids associated with any organelle, suchas the mitochondria, ribosomal RNA, and nucleic acid molecules comprisedchimerically of one or more components that are not naturally occurringalong with naturally occurring components.

Additionally, a “nucleic acid molecule” may contain in part one or morenon-nucleotide-based components as exemplified by, but not limited to,amino acids and sugars. Thus, by way of example, but not limitation, aribozyme that is in part nucleotide-based and in part protein-based isconsidered a “nucleic acid molecule”.

In addition, by way of example, but not limitation, a nucleic acidmolecule that is labeled with a detectable moiety, such as a radioactiveor alternatively a non-radioactive label, is likewise considered a“nucleic acid molecule”.

The terms “nucleic acid sequence coding for” or a “DNA coding sequenceof” or a “nucleotide sequence encoding” a particular enzyme—as well asother synonymous terms—refer to a DNA sequence which is transcribed andtranslated into an enzyme when placed under the control of appropriateregulatory sequences. A “promotor sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. The promoter is part ofthe DNA sequence. This sequence region has a start codon at its 3′terminus. The promoter sequence does include the minimum number of baseswhere elements necessary to initiate transcription at levels detectableabove background. However, after the RNA polymerase binds the sequenceand transcription is initiated at the start codon (3′ terminus with apromoter), transcription proceeds downstream in the 3′ direction. Withinthe promotor sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease S1) as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The terms “nucleic acid encoding an enzyme (protein)” or “DNA encodingan enzyme (protein)” or “polynucleotide encoding an enzyme (protein)”and other synonymous terms encompasses a polynucleotide which includesonly coding sequence for the enzyme as well as a polynucleotide whichincludes additional coding and/or non-coding sequence.

In one preferred embodiment, a “specific nucleic acid molecule species”is defined by its chemical structure, as exemplified by, but not limitedto, its primary sequence. In another preferred embodiment, a specific“nucleic acid molecule species” is defined by a function of the nucleicacid species or by a function of a product derived from the nucleic acidspecies. Thus, by way of non-limiting example, a “specific nucleic acidmolecule species” may be defined by one or more activities or propertiesattributable to it, including activities or properties attributable itsexpressed product.

The instant definition of “assembling a working nucleic acid sample intoa nucleic acid library” includes the process of incorporating a nucleicacid sample into a vector-based collection, such as by ligation into avector and transformation of a host. A description of relevant vectors,hosts, and other reagents as well as specific non-limiting examplesthereof are provided hereinafter. The instant definition of “assemblinga working nucleic acid sample into a nucleic acid library” also includesthe process of incorporating a nucleic acid sample into anon-vector-based collection, such as by ligation to adaptors. Preferablythe adaptors can anneal to PCR primers to facilitate amplification byPCR.

Accordingly, in a non-limiting embodiment, a “nucleic acid library” iscomprised of a vector-based collection of one or more nucleic acidmolecules. In another preferred embodiment a “nucleic acid library” iscomprised of a non-vector-based collection of nucleic acid molecules. Inyet another preferred embodiment a “nucleic acid library” is comprisedof a combined collection of nucleic acid molecules that is in partvector-based and in part non-vector-based. Preferably, the collection ofmolecules comprising a library is searchable and separable according toindividual nucleic acid molecule species.

The present invention provides a “nucleic acid construct” oralternatively a “nucleotide construct” or alternatively a “DNAconstruct”. The term “construct” is used herein to describe a molecule,such as a polynucleotide (e.g., a phytase polynucleotide) may optionallybe chemically bonded to one or more additional molecular moieties, suchas a vector, or parts of a vector. In a specific—but by no meanslimiting—aspect, a nucleotide construct is exemplified by a DNAexpression DNA expression constructs suitable for the transformation ofa host cell.

An “oligonucleotide” (or synonymously an “oligo”) refers to either asingle stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides may or may not have a 5′ phosphate. Thosethat do not will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleofide will ligate to a fragment that has not beendephosphorylated. To achieve polymerase-based amplification (such aswith PCR), a “32-fold degenerate oligonucleotide that is comprised of,in series, at least a first homologous sequence, a degenerate N,N,G/Tsequence, and a second homologous sequence” is mentioned. As used inthis context, “homologous” is in reference to homology between the oligoand the parental polynucleotide that is subjected to thepolymerase-based amplification.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlemRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable cultured yeast cell ormammalian cell. For example, the intracellular conditions in a yeastcell grown under typical laboratory culture conditions are physiologicalconditions. Suitable in vitro reaction conditions for in vitrotranscription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45° C. and 0.001-10 mM divalent cation (e.g., Mg⁺⁺,Ca⁺⁺); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalentcation, and often include 0.01-1.0 percent nonspecific protein (e.g.,BSA). A non-ionic detergent (Tween, NP40, Triton X-100) can often bepresent, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or non-ionic detergents and/or membrane fractionsand/or anti-foam agents and/or scintillants.

The term “population” as used herein means a collection of componentssuch as polynucleotides, portions or polynucleotides or proteins. A“mixed population: means a collection of components which belong to thesame family of nucleic acids or proteins (i.e., are related) but whichdiffer in their sequence (i.e., are not identical) and hence in theirbiological activity.

A molecule having a “pro-form” refers to a molecule that undergoes anycombination of one or more covalent and noncovalent chemicalmodifications (e.g. glycosylation, proteolytic cleavage, dimerization oroligomerization, temperature-induced or pH-induced conformationalchange, association with a co-factor, etc.) en route to attain a moremature molecular form having a property difference (e.g. an increase inactivity) in comparison with the reference pro-form molecule. When twoor more chemical modification (e.g. two proteolytic cleavages, or aproteolytic cleavage and a deglycosylation) can be distinguished enroute to the production of a mature molecule, the reference precursormolecule may be termed a “pre-pro-form” molecule.

As used herein, the term “pseudorandom” refers to a set of sequencesthat have limited variability, such that, for example, the degree ofresidue variability at another position, but any pseudorandom positionis allowed some degree of residue variation, however circumscribed.

“Quasi-repeated units”, as used herein, refers to the repeats to bere-assorted and are by definition not identical. Indeed the method isproposed not only for practically identical encoding units produced bymutagenesis of the identical starting sequence, but also thereassortment of similar or related sequences which may divergesignificantly in some regions. Nevertheless, if the sequences containsufficient homologies to be reassorted by this approach, they can bereferred to as “quasi-repeated” units.

As used herein “random peptide library” refers to a set ofpolynucleotide sequences that encodes a set of random peptides, and tothe set of random peptides encoded by those polynucleotide sequences, aswell as the fusion proteins contain those random peptides.

As used herein, “random peptide sequence” refers to an amino acidsequence composed of two or more amino acid monomers and constructed bya stochastic or random process. A random peptide can include frameworkor scaffolding motifs, which may comprise invariant sequences.

As used herein, “receptor” refers to a molecule that has an affinity fora given ligand. Receptors can be naturally occurring or syntheticmolecules. Receptors can be employed in an unaltered state or asaggregates with other species. Receptors can be attached, covalently ornon-covalently, to a binding member, either directly or via a specificbinding substance. Examples of receptors include, but are not limitedto, antibodies, including monoclonal antibodies and antisera reactivewith specific antigenic determinants (such as on viruses, cells, orother materials), cell membrane receptors, complex carbohydrates andglycoproteins, enzymes, and hormone receptors.

“Recombinant” enzymes refer to enzymes produced by recombinant DNAtechniques, i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired enzyme. “Synthetic” enzymes are thoseprepared by chemical synthesis.

The term “related polynucleotides” means that regions or areas of thepolynucleotides are identical and regions or areas of thepolynucleotides are heterologous.

“Reductive reassortment”, as used herein, refers to the increase inmolecular diversity that is accrued through deletion (and/or insertion)events that are mediated by repeated sequences.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence,” “comparisonwindow,” “sequence identity,” “percentage of sequence identity,” and“substantial identity.”

A “reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the two polynucleotidesand (2) may further comprise a sequence that is divergent between thetwo polynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity.

“Repetitive Index (RI)”, as used herein, is the average number of copiesof the quasi-repeated units contained in the cloning vector.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. This “substantial identity”, as used herein,denotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence having at least 80 percent sequenceidentity, preferably at least 85 percent identity, often 90 to 95percent sequence identity, and most commonly at least 99 percentsequence identity as compared to a reference sequence of a comparisonwindow of at least 25-50 nucleotides, wherein the percentage of sequenceidentity is calculated by comparing the reference sequence to thepolynucleotide sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the window ofcomparison.

As known in the art “similarity” between two enzymes is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one enzyme to the sequence of a second enzyme. Similaritymay be determined by procedures which are well-known in the art, forexample, a BLAST program (Basic Local Alignment Search Tool at theNational Center for Biological Information).

As used herein, the term “single-chain antibody” refers to a polypeptidecomprising a V_(H) domain and a V_(L) domain in polypeptide linkage,generally liked via a spacer peptide (e.g., (Gly-Gly-Gly-Gly-Ser_(x)),and which may comprise additional amino acid sequences at the amino-and/or carboxy-termini. For example, a single-chain antibody maycomprise a tether segment for linking to the encoding polynucleotide. Asan example, a scFv is a single-chain antibody. Single-chain antibodiesare generally proteins consisting of one or more polypeptide segments ofat least 10 contiguous amino substantially encoded by genes of theimmunoglobulin superfamily (e.g., see Williams and Barclay, 1989, pp.361-368, which is incorporated herein by reference), most frequentlyencoded by a rodent, non-human primate, avian, porcine bovine, ovine,goat, or human heavy chain or light chain gene sequence. A functionalsingle-chain antibody generally contains a sufficient portion of animmunoglobulin superfamily gene product so as to retain the property ofbinding to a specific target molecule, typically a receptor or antigen(epitope).

The members of a pair of molecules (e.g., an antibody-antigen pair or anucleic acid pair) are said to “specifically bind” to each other if theybind to each other with greater affinity than to other, non-specificmolecules. For example, an antibody raised against an antigen to whichit binds more efficiently than to a non-specific protein can bedescribed as specifically binding to the antigen. (Similarly, a nucleicacid probe can be described as specifically binding to a nucleic acidtarget if it forms a specific duplex with the target by base pairinginteractions (see above).)

“Specific hybridization” is defined herein as the formation of hybridsbetween a first polynucleotide and a second polynucleotide (e.g., apolynucleotide having a distinct but substantially identical sequence tothe first polynucleotide), wherein substantially unrelatedpolynucleotide sequences do not form hybrids in the mixture.

The term “specific polynucleotide” means a polynucleotide having certainend points and having a certain nucleic acid sequence. Twopolynucleotides wherein one polynucleotide has the identical sequence asa portion of the second polynucleotide but different ends comprises twodifferent specific polynucleotides.

“Stringent hybridization conditions” means hybridization will occur onlyif there is at least 90% identity, preferably at least 95% identity andmost preferably at least 97% identity between the sequences. SeeSambrook et al, 1989, which is hereby incorporated by reference in itsentirety.

Also included in the invention are polypeptides having sequences thatare “substantially identical” to the sequence of a phytase polypeptide,such as one of SEQ ID 1. A “substantially identical” amino acid sequenceis a sequence that differs from a reference sequence only byconservative amino acid substitutions, for example, substitutions of oneamino acid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid, or glutamine for asparagine).

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence or by one or morenon-conservative substitutions, deletions, or insertions, particularlywhen such a substitution occurs at a site that is not the active sitethe molecule, and provided that the polypeptide essentially retains itsbehavioural properties. For example, one or more amino acids can bedeleted from a phytase polypeptide, resulting in modification of thestructure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for phytase biological activity can beremoved. Such modifications can result in the development of smalleractive phytase polypeptides.

The present invention provides a “substantially pure enzyme”. The term“substantially pure enzyme” is used herein to describe a molecule, suchas a polypeptide (e.g., a phytase polypeptide, or a fragment thereof)that is substantially free of other proteins, lipids, carbohydrates,nucleic acids, and other biological materials with which it is naturallyassociated. For example, a substantially pure molecule, such as apolypeptide, can be at least 60%, by dry weight, the molecule ofinterest. The purity of the polypeptides can be determined usingstandard methods including, e.g., polyacrylamide gel electrophoresis(e.g., SDS-PAGE), column chromatography (e.g., high performance liquidchromatography (HPLC)), and amino-terminal amino acid sequence analysis.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual macromolecular species in the composition),and preferably substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies. Solvent species, small molecules (<500 Daltons), and elementalion species are not considered macromolecular species.

As used herein, the term “variable segment” refers to a portion of anascent peptide which comprises a random, pseudorandom, or definedkernal sequence. A variable segment” refers to a portion of a nascentpeptide which comprises a random pseudorandom, or defined kernalsequence. A variable segment can comprise both variant and invariantresidue positions, and the degree of residue variation at a variantresidue position may be limited: both options are selected at thediscretion of the practitioner. Typically, variable segments are about 5to 20 amino acid residues in length (e.g., 8 to 10), although variablesegments may be longer and may comprise antibody portions or receptorproteins, such as an antibody fragment, a nucleic acid binding protein,a receptor protein, and the like.

The term “wild-type” means that the polynucleotide does not comprise anymutations. A “wild type” protein means that the protein will be activeat a level of activity found in nature and will comprise the amino acidsequence found in nature.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein is directed to the use of repeated cyclesof reductive reassortment, recombination and selection which allow forthe directed molecular evolution of highly complex linear sequences,such as DNA, RNA or proteins thorough recombination.

In vivo shuffling of molecules can be performed utilizing the naturalproperty of cells to recombine multimers. While recombination in vivohas provided the major natural route to molecular diversity, geneticrecombination remains a relatively complex process that involves 1) therecognition of homologies; 2) strand cleavage, strand invasion, andmetabolic steps leading to the production of recombinant chiasma; andfinally 3) the resolution of chiasma into discrete recombined molecules.The formation of the chiasma requires the recognition of homologoussequences.

In a preferred embodiment, the invention relates to a method forproducing a hybrid polynucleotide from at least a first polynucleotideand a second polynucleotide. The present invention can be used toproduce a hybrid polynucleotide by introducing at least a firstpolynucleotide and a second polynucleotide which share at least oneregion of partial sequence homology into a suitable host cell. Theregions of partial sequence homology promote processes which result insequence reorganization producing a hybrid polynucleotide. The term“hybrid polynucleotide”, as used herein, is any nucleotide sequencewhich results from the method of the present invention and containssequence from at least two original polynucleotide sequences. Suchhybrid polynucleotides can result from intermolecular recombinationevents which promote sequence integration between DNA molecules. Inaddition, such hybrid polynucleotides can result from intramolecularreductive reassortment processes which utilize repeated sequences toalter a nucleotide sequence within a DNA molecule.

The invention provides a means for generating hybrid polynucleotideswhich may encode biologically active hybrid polypeptides. In one aspect,the original polynucleotides encode biologically active polypeptides.The method of the invention produces new hybrid polypeptides byutilizing cellular processes which integrate the sequence of theoriginal polynucleotides such that the resulting hybrid polynucleotideencodes a polypeptide demonstrating activities derived from the originalbiologically active polypeptides. For example, the originalpolynucleotides may encode a particular enzyme from differentmicroorganisms. An enzyme encoded by a first polynucleotide from oneorganism may, for example, function effectively under a particularenvironmental condition, e.g. high salinity. An enzyme encoded by asecond polynucleotide from a different organism may function effectivelyunder a different environmental condition, such as extremely hightemperatures. A hybrid polynucleotide containing sequences from thefirst and second original polynucleotides may encode an enzyme whichexhibits characteristics of both enzymes encoded by the originalpolynucleotides. Thus, the enzyme encoded by the hybrid polynucleotidemay function effectively under environmental conditions shared by eachof the enzymes encoded by the first and second polynucleotides, e.g.,high salinity and extreme temperatures.

Enzymes encoded by the original polynucleotides of the inventioninclude, but are not limited to; oxidoreductases, transferases,hydrolases, lyases, isomerases and ligases. A hybrid polypeptideresulting from the method of the invention may exhibit specializedenzyme activity not displayed in the original enzymes. For example,following recombination and/or reductive reassortment of polynucleotidesencoding hydrolase activities, the resulting hybrid polypeptide encodedby a hybrid polynucleotide can be screened for specialized hydrolaseactivities obtained from each of the original enzymes, i.e. the type ofbond on which the hydrolase acts and the temperature at which thehydrolase functions. Thus, for example, the hydrolase may be screened toascertain those chemical functionalities which distinguish the hybridhydrolase from the original hydrolyases, such as: (a) amide (peptidebonds), i.e. proteases; (b) ester bonds, i.e. esterases and lipases; (c)acetals, i.e., glycosidases and, for example, the temperature, pH orsalt concentration at which the hybrid polypeptide functions.

Sources of the original polynucleotides may be isolated from individualorganisms (“isolates”), collections of organisms that have been grown indefined media (“enrichment cultures”), or, most preferably, uncultivatedorganisms (“environmental samples”). The use of a culture-independentapproach to derive polynucleotides encoding novel bioactivities fromenvironmental samples is most preferable since it allows one to accessuntapped resources of biodiversity.

“Environmental libraries” are generated from environmental samples andrepresent the collective genomes of naturally occurring organismsarchived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions which promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

The microorganisms from which the polynucleotide may be prepared includeprokaryotic microorganisms, such as Eubacteria and Archaebacteria, andlower eukaryotic microorganisms such as fungi, some algae and protozoa.Polynucleotides may be isolated from environmental samples in which casethe nucleic acid may be recovered without culturing of an organism orrecovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.Polynucleotides encoding enzymes isolated from extremophilicmicroorganisms are particularly preferred. Such enzymes may function attemperatures above 100° C. in terrestrial hot springs and deep seathermal vents, at temperatures below 0° C. in arctic waters, in thesaturated salt environment of the Dead Sea, at pH values around 0 incoal deposits and geothermal sulfur-rich springs, or at pH valuesgreater than 11 in sewage sludge. For example, several esterases andlipases cloned and expressed from extremophilic organisms show highactivity throughout a wide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described areintroduced into a suitable host cell. A suitable host cell is any cellwhich is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides are preferably already in avector which includes appropriate control sequences. The host cell canbe a higher eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, or preferably, the host cell canbe a prokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis, Dibner, & Battey, 1986).

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; and plant cells. The selection of anappropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described by Gluzman, Cell, 23:175 (1981), and other celllines capable of expressing a compatible vector, for example, the C127,3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors willcomprise an origin of replication, a suitable promoter and enhancer, andalso any necessary ribosome binding sites, polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences, and 5′flanking nontranscribed sequences. DNA sequences derived from the SV40splice, and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

Host cells containing the polynucleotides of interest can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan. The clones which are identified ashaving the specified enzyme activity may then be sequenced to identifythe polynucleotide sequence encoding an enzyme having the enhancedactivity.

In another aspect, it is envisioned the method of the present inventioncan be used to generate novel polynucleotides encoding biochemicalpathways from one or more operons or gene clusters or portions thereof.For example, bacteria and many eukaryotes have a coordinated mechanismfor regulating genes whose products are involved in related processes.The genes are clustered, in structures referred to as “gene clusters,”on a single chromosome and are transcribed together under the control ofa single regulatory sequence, including a single promoter whichinitiates transcription of the entire cluster. Thus, a gene cluster is agroup of adjacent genes that are either identical or related, usually asto their function. An example of a biochemical pathway encoded by geneclusters are polyketides. Polyketides are molecules which are anextremely rich source of bioactivities, including antibiotics (such astetracyclines and erythromycin), anti-cancer agents (daunomycin),immunosuppressants (FK506 and rapamycin), and veterinary products(monensin). Many polyketides (produced by polyketide synthases) arevaluable as therapeutic agents. Polyketide synthases are multifunctionalenzymes that catalyze the biosynthesis of an enormous variety of carbonchains differing in length and patterns of functionality andcyclization. Polyketide synthase genes fall into gene clusters and atleast one type (designated type 1) of polyketide synthases have largesize genes and enzymes, complicating genetic manipulation and in vitrostudies of these genes/proteins.

The ability to select and combine desired components from a library ofpolyketides, or fragments thereof, and postpolyketide biosynthesis genesfor generation of novel polyketides for study is appealing. The methodof the present invention makes it possible to facilitate the productionof novel polyketide synthases through intermolecular recombination.

Preferably, gene cluster DNA can be isolated from different organismsand ligated into vectors, particularly vectors containing expressionregulatory sequences which can control and regulate the production of adetectable protein or protein-related array activity from the ligatedgene clusters. Use of vectors which have an exceptionally large capacityfor exogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affect high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples. Onceligated into an appropriate vector, two or more vectors containingdifferent polyketide synthase gene clusters can be introduced into asuitable host cell. Regions of partial sequence homology shared by thegene clusters will promote processes which result in sequencereorganization resulting in a hybrid gene cluster. The novel hybrid genecluster can then be screened for enhanced activities not found in theoriginal gene clusters.

Therefore, in a preferred embodiment, the present invention relates to amethod for producing a biologically active hybrid polypeptide andscreening such a polypeptide for enhanced activity by:

-   -   1) introducing at least a first polynucleotide in operable        linkage and a second polynucleotide in operable linkage, said at        least first polynucleotide and second polynucleotide sharing at        least one region of partial sequence homology, into a suitable        host cell;    -   2) growing the host cell under conditions which promote sequence        reorganization resulting in a hybrid polynucleotide in operable        linkage;    -   3) expressing a hybrid polypeptide encoded by the hybrid        polynucleotide;    -   4) screening the hybrid polypeptide under conditions which        promote identification of enhanced biological activity; and    -   5) isolating the a polynucleotide encoding the hybrid        polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and discussed throughout the present specification.Such methods may be employed when isolating the polypeptides andpolynucleotides of the present invention.

As representative examples of expression vectors which may be used theremay be mentioned viral particles, baculovirus, phage, plasmids,phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA(e.g. vaccinia, adenovirus, foul pox virus, pseudorabies and derivativesof SV40), P1-based artificial chromosomes, yeast plasmids, yeastartificial chromosomes, and any other vectors specific for specifichosts of interest (such as bacillus, aspergillus and yeast). Thus, forexample, the DNA may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences. Large numbers of suitablevectors are known to those of skill in the art, and are commerciallyavailable. The following vectors are provided by way of example;Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors,(lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T(Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG,pSVLSV40 (Pharmacia). However, any other plasmid or other vector may beused as long as they are replicable and viable in the host. Low copynumber or high copy number vectors may be employed with the presentinvention.

A preferred type of vector for use in the present invention contains anf-factor origin replication. The f-factor (or fertility factor) in E.coli is a plasmid which effects high frequency transfer of itself duringconjugation and less frequent transfer of the bacterial chromosomeitself. A particularly preferred embodiment is to use cloning vectors,referred to as “fosmids” or bacterial artificial chromosome (BAC)vectors. These are derived from E. coli f-factor which is able to stablyintegrate large segments of genomic DNA. When integrated with DNA from amixed uncultured environmental sample, this makes it possible to achievelarge genomic fragments in the form of a stable “environmental DNAlibrary.”

Another preferred type of vector for use in the present invention is acosmid vector. Cosmid vectors were originally designed to clone andpropagate large segments of genomic DNA. Cloning into cosmid vectors isdescribed in detail in Sambrook, et al., Molecular Cloning A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lacI, lacZ, T3,T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using CAT (chloramphenicoltransferase) vectors or other vectors with selectable markers.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycol)tic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium.

The cloning strategy permits expression via both vector driven andendogenous promoters; vector promotion may be important with expressionof genes whose endogenous promoter will not function in E. coli.

The DNA isolated or derived from microorganisms can preferably beinserted into a vector or a plasmid prior to probing for selected DNA.Such vectors or plasmids are preferably those containing expressionregulatory sequences, including promoters, enhancers and the like. Suchpolynucleotides can be part of a vector and/or a composition and stillbe isolated, in that such vector or composition is not part of itsnatural environment. Particularly preferred phage or plasmid and methodsfor introduction and packaging into them are described in detail in theprotocol set forth herein.

The selection of the cloning vector depends upon the approach taken, forexample, the vector can be any cloning vector with an adequate capacityfor multiply repeated copies of a sequence, or multiple sequences thatcan be successfully transformed and selected in a host cell. One exampleof such a vector is described in “Polycos vectors: a system forpackaging filamentous phage and phagemid vectors using lambda phagepackaging extracts”, Alting-Mecs M A, Short J M, Gene, 1993 Dec 27,137:1, 93-100. Propagation/maintenance can be by an antibioticresistance carried by the cloning vector. After a period of growth, thenaturally abbreviated molecules are recovered and identified by sizefractionation on a gel or column, or amplified directly. The cloningvector utilized may contain a selectable gene that is disrupted by theinsertion of the lengthy construct. As reductive reassortmentprogresses, the number of repeated units is reduced and the interruptedgene is again expressed and hence selection for the processed constructcan be applied. The vector may be an expression/selection vector whichwill allow for the selection of an expressed product possessingdesirable biologically properties. The insert may be positioneddownstream of a functional promotor and the desirable property screenedby appropriate means.

In vivo reassortment is focused on “inter-molecular” processescollectively referred to as “recombination” which in bacteria, isgenerally viewed as a “RecA-dependent” phenomenon. The present inventioncan rely on recombination processes of a host cell to recombine andre-assort sequences, or the cells' ability to mediate reductiveprocesses to decrease the complexity of quasi-repeated sequences in thecell by deletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process.

Therefore, in another aspect of the present invention, novelpolynucleotides can be generated by the process of reductivereassortment. The method involves the generation of constructscontaining consecutive sequences (original encoding sequences), theirinsertion into an appropriate vector, and their subsequent introductioninto an appropriate host cell. The reassortment of the individualmolecular identities occurs by combinatorial processes between theconsecutive sequences in the construct possessing regions of homology,or between quasi-repeated units. The reassortment process recombinesand/or reduces the complexity and extent of the repeated sequences, andresults in the production of novel molecular species. Various treatmentsmay be applied to enhance the rate of reassortment. These could includetreatment with ultra-violet light, or DNA damaging chemicals, and/or theuse of host cell lines displaying enhanced levels of “geneticinstability”. Thus the reassortment process may involve homologousrecombination or the natural property of quasi-repeated sequences todirect their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. In the present invention, “quasi-repeats” are repeats thatare not restricted to their original unit structure. Quasi-repeatedunits can be presented as an array of sequences in a construct;consecutive units of similar sequences. Once ligated, the junctionsbetween the consecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.The constructs containing the quasi-repeats thus effectively providesufficient molecular elasticity that deletion (and potentiallyinsertion) events can occur virtually anywhere within thequasi-repetitive units.

When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

-   -   a) Primers that include a poly-A head and poly-T tail which when        made single-stranded would provide orientation can be utilized.        This is accomplished by having the first few bases of the        primers made from RNA and hence easily removed RNAseH.    -   b) Primers that include unique restriction cleavage sites can be        utilized. Multiple sites, a battery of unique sequences, and        repeated synthesis and ligation steps would be required.    -   c) The inner few bases of the primer could be thiolated and an        exonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced RI. The re-assorted encoding sequencescan then be recovered by amplification. The products are re-cloned andexpressed. The recovery of cloning vectors with reduced RI can beeffected by:

-   -   1) The use of vectors only stably maintained when the construct        is reduced in complexity.    -   2) The physical recovery of shortened vectors by physical        procedures. In this case, the cloning vector would be recovered        using standard plasmid isolation procedures and size        fractionated on either an agarose gel, or column with a low        molecular weight cut off utilizing standard procedures.    -   3) The recovery of vectors containing interrupted genes which        can be selected when insert size decreases.    -   4) The use of direct selection techniques with an expression        vector and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

The following example demonstrates the method of the invention. Encodingnucleic acid sequences (quasi-repeats) derived from three (3) uniquespecies are depicted. Each sequence encodes a protein with a distinctset of properties. Each of the sequences differs by a single or a fewbase pairs at a unique position in the sequence which are designated“A”, “B” and “C”. The quasi-repeated sequences are separately orcollectively amplified and ligated into random assemblies such that allpossible permutations and combinations are available in the populationof ligated molecules. The number of quasi-repeat units can be controlledby the assembly conditions. The average number of quasi-repeated unitsin a construct is defined as the repetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector, and transfected into an appropriate host cell. The cells arethen propagated and “reductive reassortment” is effected. The rate ofthe reductive reassortment process may be stimulated by the introductionof DNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

Optionally, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact (e.g.,such as catalytic antibodies) with a predetermined macromolecule, suchas for example a proteinaceous receptor, peptide oligosaccharide, viron,or other predetermined compound or structure.

The displayed polypeptides, antibodies, peptidomimetic antibodies, andvariable region sequences that are identified from such libraries can beused for therapeutic, diagnostic, research and related purposes (e.g.,catalysts, solutes for increasing osmolarity of an aqueous solution, andthe like), and/or can be subjected to one or more additional cycles ofshuffling and/or affinity selection. The method can be modified suchthat the step of selecting for a phenotypic characteristic can be otherthan of binding affinity for a predetermined molecule (e.g., forcatalytic activity, stability oxidation resistance, drug resistance, ordetectable phenotype conferred upon a host cell).

The present invention provides a method for generating libraries ofdisplayed antibodies suitable for affinity interactions screening. Themethod comprises (1) obtaining first a plurality of selected librarymembers comprising a displayed antibody and an associated polynucleotideencoding said displayed antibody, and obtaining said associatedpolynucleotide encoding for said displayed antibody and obtaining saidassociated polynucleotides or copies thereof, wherein said associatedpolynucleotides comprise a region of substantially identical variableregion framework sequence, and (2) introducing said polynucleotides intoa suitable host cell and growing the cells under conditions whichpromote recombination and reductive reassortment resulting in shuffledpolynucleotides. CDR combinations comprised by the shuffled pool are notpresent in the first plurality of selected library members, saidshuffled pool composing a library of displayed antibodies comprising CDRpermutations and suitable for affinity interaction screening.Optionally, the shuffled pool is subjected to affinity screening toselect shuffled library members which bind to a predetermined epitope(antigen) and thereby selecting a plurality of selected shuffled librarymembers. Further, the plurality of selectively shuffled library memberscan be shuffled and screened iteratively, from 1 to about 1000 cycles oras desired until library members having a desired binding affinity areobtained.

In another aspect of the invention, it is envisioned that prior to orduring recombination or reassortment, polynucleotides generated by themethod of the present invention can be subjected to agents or processeswhich promote the introduction of mutations into the originalpolynucleotides. The introduction of such mutations would increase thediversity of resulting hybrid polynucleotides and polypeptides encodedtherefrom. The agents or processes which promote mutagenesis caninclude, but are not limited to: (+)-CC-1065, or a synthetic analog suchas (+)-CC-1065-(N3-Adenine), (see. Biochem. 31, 2822-2829 (1992)); aN-acelylated or deacetylated 4′-fluro-4-aminobiphenyl adduct capable ofinhibiting DNA synthesis (see, for example, Carcinogenesis vol. 13, No.5, 751-758 (1992); or a N-acetylated or deacetylated 4-aminobiphenyladduct capable of inhibiting DNA synthesis (see also, Id. 751-758);trivalent chromium, a trivalent chromium salt, a polycyclic aromatichydrocarbon (“PAH”) DNA adduct capable of inhibiting DNA replication,such as 7-bromomethyl-benz[a]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”), andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Especially preferred “means for slowing or haltingPCR amplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N3-Adenine). Particularly encompassed means are DNA adductsor polynucleotides comprising the DNA adducts from the polynucleotidesor polynucleotides pool, which can be released or removed by a processincluding heating the solution comprising the polynucleotides prior tofurther processing.

In another aspect the present invention is directed to a method ofproducing recombinant proteins having biological activity by treating asample comprising double-stranded template polynucleotides encoding awild-type protein under conditions according to the present inventionwhich provide for the production of hybrid or re-assortedpolynucleotides.

The invention also provides the use of polynucleotide shuffling toshuffle a population of viral genes (e.g., capsid proteins, spikeglycoproteins, polymerases, and proteases) or viral genomes (e.g.,paramyxoviridae, orthomyxoviridae, herpesviruses, retroviruses,reoviruses and rhinoviruses). In an embodiment, the invention provides amethod for shuffling sequences encoding all or portions of immunogenicviral proteins to generate novel combinations of epitopes as well asnovel epitopes created by recombination; such shuffled viral proteinsmay comprise epitopes or combinations of epitopes as well as novelepitopes created by recombination; such shuffled viral proteins maycomprise epitopes or combinations of epitopes which are likely to arisein the natural environment as a consequence of viral evolution; (e.g.,such as recombination of influenza virus strains).

The invention also provides a method suitable for shufflingpolynucleotide sequences for generating gene therapy vectors andreplication-defective gene therapy constructs, such as may be used forhuman gene therapy, including but not limited to vaccination vectors forDNA-based vaccination, as well as anti-neoplastic gene therapy and othergeneral therapy formats.

In the polypeptide notation used herein, the left-hand direction is theamino terminal direction and the right-hand direction is thecarboxy-terminal direction, in accordance with standard usage andconvention. Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the coding RNA transcriptare referred to as “downstream sequences”.

Methodology

Nucleic acid shuffling is a method for in vitro or in vivo homologousrecombination of pools of shorter or smaller polynucleotides to producea polynucleotide or polynucleotides. Mixtures of related nucleic acidsequences or polynucleotides are subjected to sexual PCR to providerandom polynucleotides, and reassembled to yield a library or mixedpopulation of recombinant hybrid nucleic acid molecules orpolynucleotides.

In contrast to cassette mutagenesis, only shuffling and error-prone PCRallow one to mutate a pool of sequences blindly (without sequenceinformation other than primers).

The advantage of the mutagenic shuffling of this invention overerror-prone PCR alone for repeated selection can best be explained withan example from antibody engineering. Consider DNA shuffling as comparedwith error-prone PCR (not sexual PCR). The initial library of selectedpooled sequences can consist of related sequences of diverse origin (i.e. antibodies from naive mRNA) or can be derived by any type ofmutagenesis (including shuffling) of a single antibody gene. Acollection of selected complementarity determining regions (“CDRs”) isobtained after the first round of affinity selection. In the diagram thethick CDRs confer onto the antibody molecule increased affinity for theantigen. Shuffling allows the free combinatorial association of all ofthe CDR1s with all of the CDR2s with all of the CDR3s, for example.

This method differs from error-prone PCR, in that it is an inverse chainreaction. In error-prone PCR, the number of polymerase start sites andthe number of molecules grows exponentially. However, the sequence ofthe polymerase start sites and the sequence of the molecules remainsessentially the same. In contrast, in nucleic acid reassembly orshuffling of random polynucleotides the number of start sites and thenumber (but not size) of the random polynucleotides decreases over time.For polynucleotides derived from whole plasmids the theoretical endpointis a single, large concatemeric molecule.

Since cross-overs occur at regions of homology, recombination willprimarily occur between members of the same sequence family. Thisdiscourages combinations of CDRs that are grossly incompatible (e.g.,directed against different epitopes of the same antigen). It iscontemplated that multiple families of sequences can be shuffled in thesame reaction. Further, shuffling generally conserves the relativeorder, such that, for example, CDR1 will not be found in the position ofCDR2.

Rare shufflants will contain a large number of the best (eg. highestaffinity) CDRs and these rare shufflants may be selected based on theirsuperior affinity.

CDRs from a pool of 100 different selected antibody sequences can bepermutated in up to 1006 different ways. This large number ofpermutations cannot be represented in a single library of DNA sequences.Accordingly, it is contemplated that multiple cycles of DNA shufflingand selection may be required depending on the length of the sequenceand the sequence diversity desired.

Error-prone PCR, in contrast, keeps all the selected CDRs in the samerelative sequence, generating a much smaller mutant cloud.

The template polynucleotide which may be used in the methods of thisinvention may be DNA or RNA. It may be of various lengths depending onthe size of the gene or shorter or smaller polynucleotide to berecombined or reassembled. Preferably, the template polynucleotide isfrom 50 bp to 50 kb. It is contemplated that entire vectors containingthe nucleic acid encoding the protein of interest can be used in themethods of this invention, and in fact have been successfully used.

The template polynucleotide may be obtained by amplification using thePCR reaction (U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,683,195) orother amplification or cloning methods. However, the removal of freeprimers from the PCR products before subjecting them to pooling of thePCR products and sexual PCR may provide more efficient results. Failureto adequately remove the primers from the original pool before sexualPCR can lead to a low frequency of crossover clones.

The template polynucleotide often should be double-stranded. Adouble-stranded nucleic acid molecule is recommended to ensure thatregions of the resulting single-stranded polynucleotides arecomplementary to each other and thus can hybridize to form adouble-stranded molecule.

It is contemplated that single-stranded or double-stranded nucleic acidpolynucleotides having regions of identity to the templatepolynucleotide and regions of heterology to the template polynucleotidemay be added to the template polynucleotide, at this step. It is alsocontemplated that two different but related polynucleotide templates canbe mixed at this step.

The double-stranded polynucleotide template and any added double-orsingle-stranded polynucleotides are subjected to sexual PCR whichincludes slowing or halting to provide a mixture of from about 5 bp to 5kb or more. Preferably the size of the random polynucleotides is fromabout 10 bp to 1000 bp, more preferably the size of the polynucleotidesis from about 20 bp to 500 bp.

Alternatively, it is also contemplated that double-stranded nucleic acidhaving multiple nicks may be used in the methods of this invention. Anick is a break in one strand of the double-stranded nucleic acid. Thedistance between such nicks is preferably 5 bp to 5 kb, more preferablybetween 10 bp to 1000 bp. This can provide areas of self-priming toproduce shorter or smaller polynucleotides to be included with thepolynucleotides resulting from random primers, for example.

The concentration of any one specific polynucleotide will not be greaterthan 1% by weight of the total polynucleotides, more preferably theconcentration of any one specific nucleic acid sequence will not begreater than 0.1% by weight of the total nucleic acid.

The number of different specific polynucletides in the mixture will beat least about 100, preferably at least about 500, and more preferablyat least about 1000.

At this step single-stranded or double-stranded polynucleotides, eithersynthetic or natural, may be added to the random double-stranded shorteror smaller polynucleotides in order to increase the heterogeneity of themixture of polynucleotides.

It is also contemplated that populations of double-stranded randomlybroken polynucleotides may be mixed or combined at this step with thepolynucleotides from the sexual PCR process and optionally subjected toone or more additional sexual PCR cycles.

Where insertion of mutations into the template polynucleotide isdesired, single-stranded or double-stranded polynucleotides having aregion of identity to the template polynucleotide and a region ofheterology to the template polynucleotide maybe added in a 20 foldexcess by weight as compared to the total nucleic acid, more preferablythe single-stranded polynucleotides may be added in a 10 fold excess byweight as compared to the total nucleic acid.

Where a mixture of different but related template polynucleotides isdesired, populations of polynucleotides from each of the templates maybe combined at a ratio of less than about 1:100, more preferably theratio is less than about 1:40. For example, a backcross of the wild-typepolynucleotide with a population of mutated polynucleotide may bedesired to eliminate neutral mutations (e.g., mutations yielding aninsubstantial alteration in the phenotypic property being selected for).In such an example, the ratio of randomly provided wild-typepolynucleotides which may be added to the randomly provided sexual PCRcycle hybrid polynucleotides is approximately 1:1 to about 100:1, andmore preferably from 1:1 to 40:1.

The mixed population of random polynucleotides are denatured to formsingle-stranded polynucleotides and then re-annealed. Only thosesingle-stranded polynucleotides having regions of homology with othersingle-stranded polynucleotides will re-anneal.

The random polynucleotides may be denatured by heating. One skilled inthe art could determine the conditions necessary to completely denaturethe double-stranded nucleic acid. Preferably the temperature is from 80°C. to 100° C., more preferably the temperature is from 90° C. to 96° C.other methods which may be used to denature the polynucleotides includepressure (36) and pH.

The polynucleotides may be re-annealed by cooling. Preferably thetemperature is from 20° C. to 75° C., more preferably the temperature isfrom 40° C. to 65° C. If a high frequency of crossovers is needed basedon an average of only 4 consecutive bases of homology, recombination canbe forced by using a low annealing temperature, although the processbecomes more difficult. The degree of renaturation which occurs willdepend on the degree of homology between the population ofsingle-stranded polynucleotides.

Renaturation can be accelerated by the addition of polyethylene glycol(“PEG”) or salt. The salt concentration is preferably from 0 mM to 200mM, more preferably the salt concentration is from 10 mM to 100 mm. Thesalt may be KCl or NaCl. The concentration of PEG is preferably from 0%to 20%, more preferably from 5% to 10%.

The annealed polynucleotides are next incubated in the presence of anucleic acid polymerase and dNTP's (i.e. dATP, dCTP, DGTP and dTTP). Thenucleic acid polymerase may be the Klenow fragment, the Taq polymeraseor any other DNA polymerase known in the art

The approach to be used for the assembly depends on the minimum degreeof homology that should still yield crossovers. If the areas of identityare large, Taq polymerase can be used with an annealing temperature ofbetween 45-65° C. If the areas of identity are small, Klenow polymerasecan be used with an annealing temperature of between 20-30° C. Oneskilled in the art could vary the temperature of annealing to increasethe number of cross-overs achieved.

The polymerase may be added to the random polynucleotides prior toannealing, simultaneously with annealing or after annealing.

The cycle of denaturation, renaturation and incubation in the presenceof polymerase is referred to herein as shuffling or reassembly of thenucleic acid. This cycle is repeated for a desired number of times.Preferably the cycle is repeated from 2 to 50 times, more preferably thesequence is repeated from 10 to 40 times.

The resulting nucleic acid is a larger double-stranded polynucleotide offrom about 50 bp to about 100 kb, preferably the larger polynucleotideis from 500 bp to 50 kb.

This larger polynucleotides may contain a number of copies of apolynucleotide having the same size as the template polynucleotide intandem. This concatemeric polynucleotide is then denatured into singlecopies of the template polynucleotide. The result will be a populationof polynucleotides of approximately the same size as the templatepolynucleotide. The population will be a mixed population where singleor double-stranded polynucleotides having an area of identity and anarea of heterology have been added to the template polynucleotide priorto shuffling.

These polynucleotides are then cloned into the appropriate vector andthe ligation mixture used to transform bacteria.

It is contemplated that the single polynucleotides may be obtained fromthe larger concatemeric polynucleotide by amplification of the singlepolynucleotide prior to cloning by a variety of methods including PCR(U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202), rather than bydigestion of the concatemer.

The vector used for cloning is not critical provided that it will accepta polynucleotide of the desired size. If expression of the particularpolynucleotide is desired, the cloning vehicle should further comprisetranscription and translation signals next to the site of insertion ofthe polynucleotide to allow expression of the polynucleotide in the hostcell. Preferred vectors include the pUC series and the pBR series ofplasmids.

The resulting bacterial population will include a number of recombinantpolynucleotides having random mutations. This mixed population may betested to identify the desired recombinant polynucleotides. The methodof selection will depend on the polynucleotide desired.

For example, if a polynucleotide which encodes a protein with increasedbinding efficiency to a ligand is desired, the proteins expressed byeach of the portions of the polynucleotides in the population or librarymay be tested for their ability to bind to the ligand by methods knownin the art (i.e. panning, affinity chromatography). If a polynucleotidewhich encodes for a protein with increased drug resistance is desired,the proteins expressed by each of the polynucleotides in the populationor library may be tested for their ability to confer drug resistance tothe host organism. One skilled in the art, given knowledge of thedesired protein, could readily test the population to identifypolynucleotides which confer the desired properties onto the protein.

It is contemplated that one skilled in the art could use a phage displaysystem in which fragments of the protein are expressed as fusionproteins on the phage surface (Pharmacia, Milwaukee Wis.). Therecombinant DNA molecules are cloned into the phage DNA at a site whichresults in the transcription of a fusion protein a portion of which isencoded by the recombinant DNA molecule. The phage containing therecombinant nucleic acid molecule undergoes replication andtranscription in the cell. The leader sequence of the fusion proteindirects the transport of the fusion protein to the tip of the phageparticle. Thus the fusion protein which is partially encoded by therecombinant DNA molecule is displayed on the phage particle fordetection and selection by the methods described above.

It is further contemplated that a number of cycles of nucleic acidshuffling may be conducted with polynucleotides from a sub-population ofthe first population, which sub-population contains DNA encoding thedesired recombinant protein. In this manner, proteins with even higherbinding affinities or enzymatic activity could be achieved.

It is also contemplated that a number of cycles of nucleic acidshuffling may be conducted with a mixture of wild-type polynucleotidesand a sub-population of nucleic acid from the first or subsequent roundsof nucleic acid shuffling in order to remove any silent mutations fromthe sub-population.

Any source of nucleic acid, in purified form can be utilized as thestarting nucleic acid. Thus the process may employ DNA or RNA includingmessenger RNA, which DNA or RNA may be single or double stranded. Inaddition, a DNA-RNA hybrid which contains one strand of each may beutilized. The nucleic acid sequence may be of various lengths dependingon the size of the nucleic acid sequence to be mutated. Preferably thespecific,nucleic acid sequence is from 50 to 50000 base pairs. It iscontemplated that entire vectors containing the nucleic acid encodingthe protein of interest may be used in the methods of this invention.

The nucleic acid may be obtained from any source, for example, fromplasmids such a pBR322, from cloned DNA or RNA or from natural DNA orRNA from any source including bacteria, yeast, viruses and higherorganisms such as plants or animals. DNA or RNA may be extracted fromblood or tissue material. The template polynucleotide may be obtained byamplification using the polynucleotide chain reaction (PCR) (U.S. Pat.No. 4,683,202 and U.S. Pat. No. 4,683,195). Alternatively, thepolynucleotide may be present in a vector present in a cell andsufficient nucleic acid may be obtained by culturing the cell andextracting the nucleic acid from the cell by methods known in the art.

Any specific nucleic acid sequence can be used to produce the populationof hybrids by the present process. It is only necessary that a smallpopulation of hybrid sequences of the specific nucleic acid sequenceexist or be created prior to the present process.

The initial small population of the specific nucleic acid sequenceshaving mutations may be created by a number of different methods.Mutations may be created by error-prone PCR. Error-prone PCR useslow-fidelity polymerization conditions to introduce a low level of pointmutations randomly over a long sequence. Alternatively, mutations can beintroduced into the template polynucleotide by oligonucleotide-directedmutagenesis. In oligonucleotide-directed mutagenesis, a short sequenceof the polynucleotide is removed from the polynucleotide usingrestriction enzyme digestion and is replaced with a syntheticpolynucleotide in which various bases have been altered from theoriginal sequence. The polynucleotide sequence can also be altered bychemical mutagenesis. Chemical mutagens include, for example, sodiumbisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. otheragents which are analogues of nucleotide precursors includenitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally,these agents are added to the PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.Random mutagenesis of the polynucleotide sequence can also be achievedby irradiation with X-rays or ultraviolet light. Generally, plasmidpolynucleotides so mutagenized are introduced into E. coli andpropagated as a pool or library of hybrid plasmids.

Alternatively the small mixed population of specific nucleic acids maybe found in nature in that they may consist of different alleles of thesame gene or the same gene from different related species (i.e., cognategenes). Alternatively, they may be related DNA sequences found withinone species, for example, the immunoglobulin genes.

Once the mixed population of the specific nucleic acid sequences isgenerated, the polynucleotides can be used directly or inserted into anappropriate cloning vector, using techniques well-known in, the art.

The choice of vector depends on the size of the polynucleotide sequenceand the host cell to be employed in the methods of this invention. Thetemplates of this invention may be plasmids, phages, cosmids, phagemids,viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses,reoviruses, paramyxoviruses, and the like), or selected portions thereof(e.g., coat protein, spike glycoprotein, capsid protein). For example,cosmids and phagemids are preferred where the specific nucleic acidsequence to be mutated is larger because these vectors are able tostably propagate large polynucleotides.

If the mixed population of the specific nucleic acid sequence is clonedinto a vector it can be clonally amplified by inserting each vector intoa host cell and allowing the host cell to amplify the vector. This isreferred to as clonal amplification because while the absolute number ofnucleic acid sequences increases, the number of hybrids does notincrease. Utility can be readily determined by screening expressedpolypeptides.

The DNA shuffling method of this invention can be performed blindly on apool of unknown sequences. By adding to the reassembly mixtureoligonucleotides (with ends that are homologous to the sequences beingreassembled) any sequence mixture can be incorporated at any specificposition into another sequence mixture. Thus, it is contemplated thatmixtures of synthetic oligonucleotides, PCR polynucleotides or evenwhole genes can be mixed into another sequence library at definedpositions. The insertion of one sequence (mixture) is independent fromthe insertion of a sequence in another part of the template. Thus, thedegree of recombination, the homology required, and the diversity of thelibrary can be independently and simultaneously varied along the lengthof the reassembled DNA.

This approach of mixing two genes may be useful for the humanization ofantibodies from murine hybridomas. The approach of mixing two genes orinserting alternative sequences into genes may be useful for anytherapeutically used protein, for example, interleukin I, antibodies,tPA and growth hormone. The approach may also be useful in any nucleicacid for example, promoters or introns or 31 untranslated region or 51untranslated regions of genes to increase expression or alterspecificity of expression of proteins. The approach may also be used tomutate ribozymes or aptamers.

Shuffling requires the presence of homologous regions separating regionsof diversity. Scaffold-like protein structures may be particularlysuitable for shuffling. The conserved scaffold determines the overallfolding by self-association, while displaying relatively unrestrictedloops that mediate the specific binding. Examples of such scaffolds arethe immunoglobulin beta-barrel, and the four-helix bundle which arewell-known in the art. This shuffling can be used to createscaffold-like proteins with various combinations of mutated sequencesfor binding.

Saturation Mutagenesis

In one aspect, this invention provides for the use of proprietary codonprimers (containing a degenerate N,N,G/T sequence) to introduce pointmutations into a polynucleotide, so as to generate a set of progenypolypeptides in which a full range of single amino acid substitutions isrepresented at each amino acid position. The oligos used are comprisedcontiguously of a first homologous sequence, a degenerate N,N,G/Tsequence, and preferably but not necessarily a second homologoussequence. The downstream progeny translational products from the use ofsuch oligos include all possible amino acid changes at each amino acidsite along the polypeptide, because the degeneracy of the N,N,G/Tsequence includes codons for all 20 amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,G/T cassette) is used for subjecting each original codon in aparental polynucleotide template to a full range of codon substitutions.In another aspect, at least two degenerate N,N,G/T cassettes areused—either in the same oligo or not, for subjecting at least twooriginal codons in a parental polynucleotide template to a full range ofcodon substitutions. Thus, more than one N,N,G/T sequence can becontained in one oligo to introduce amino acid mutations at more thanone site. This plurality of N,N,G/T sequences can be directlycontiguous, or separated by one or more additional nucleotidesequence(s). In another aspect, oligos serviceable for introducingadditions and deletions can be used either alone or in combination withthe codons containing an N,N,G/T sequence, to introduce any combinationor permutation of amino acid additions, deletions, and/or substitutions.

In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)nsequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,G/T sequence.For example, it may be desirable in some instances to use (e.g. in anoligo) a degenerate triplet sequence comprised of only one N, where saidN can be in the first second or third position of the triplet. Any otherbases including any combinations and permutations thereof can be used inthe remaining two postitions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g. in an oligo) a degenerate N,N,Ntriplet sequence.

It is appreciated, however, that the use of a degenerate N,N,G/T tripletas disclosed in the instant invention is advantageous for severalreasons. In one aspect, this invention provides a means tosystematically and fairly easily generate the substitution of the fullrange of possible amino acids (for a total of 20 amino acids) into eachand every amino acid position in a polypeptide. Thus, for a 100 aminoacid polypeptide, the instant invention provides a way to systematicallyand fairly easily generate 2000 distinct species (i.e. 20 possible aminoacids per position ×100 amino acid positions). It is appreciated thatthere is provided, through the use of an oligo containing a degenerateN,N,G/T triplet, 32 individual sequences that code for 20 possible aminoacids. Thus, in a reaction vessel in which a parental polynucleotidesequence is subjected to saturation mutagenesis using one such oligo,there are generated 32 distinct progeny polynucleotides encoding 20distinct polypeptides. In contrast, the use of a non-degenerate oligo insite-directed mutagenesis leads to only one progeny polypeptide productper reaction vessel.

Thus, in a preferred embodiment of this invention, each saturationmutagenesis reaction vessel contains polynucleotides encoding at least20 progeny polypeptide molecules such that all 20 amino acids arerepresented at the one specific amino acid position corresponding to thecodon position mutagenized in the parental polynucleotide. The 32-folddegenerate progeny polypeptides generated from each saturationmutagenesis reaction vessel can be subjected to clonal amplification(e.g. cloned into a suitable E. coli host using an expression vector)and subjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid, and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined—6 single pointmutations (i.e. 2 at each of three positions) and no change at anyposition.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

Thus, in a non-limiting exemplification, this invention provides for theuse of saturation mutagenesis in combination with additionalmutagenization processes, such as process where two or more relatedpolynucleotides are introduced into a suitable host cell such that ahybrid polynucleotide is generated by recombination and reductivereassortment.

In Vitro Shuffling

The equivalents of some standard genetic matings may also be performedby shuffling in vitro. For example, a “molecular backcross” can beperformed by repeatedly mixing the hybrid's nucleic acid with thewild-type nucleic acid while selecting for the mutations of interest. Asin traditional breeding, this approach can be used to combine phenotypesfrom different sources into a background of choice. It is useful, forexample, for the removal of neutral mutations that affect unselectedcharacteristics (i.e. immunogenicity). Thus it can be useful todetermine which mutations in a protein are involved in the enhancedbiological activity and which are not, an advantage which cannot beachieved by error-prone mutagenesis or cassette mutagenesis methods.

Large, functional genes can be assembled correctly from a mixture ofsmall random polynucleotides. This reaction may be of use for thereassembly of genes from the highly fragmented DNA of fossils. Inaddition random nucleic acid fragments from fossils may be combined withpolynucleotides from similar genes from related species.

It is also contemplated that the method of this invention can be usedfor the in vitro amplification of a whole genome from a single cell asis needed for a variety of research and diagnostic applications. DNAamplification by PCR is in practice limited to a length of about 40 kb.Amplification of a whole genome such as that of E. coli (5, 000 kb) byPCR would require about 250 primers yielding 125 forty kbpolynucleotides. This approach is not practical due to theunavailability of sufficient sequence data. On the other hand, randomproduction of polynucleotides of the genome with sexual PCR cycles,followed by gel purification of small polynucleotides will provide amultitude of possible primers. Use of this mix of random smallpolynucleotides as primers in a PCR reaction alone or with the wholegenome as the template should result in an inverse chain reaction withthe theoretical endpoint of a single concatemer containing many copiesof the genome.

100 fold amplification in the copy number and an average polynucleotidesize of greater than 50 kb may be obtained when only randompolynucleotides are used. It is thought that the larger concatemer isgenerated by overlap of many smaller polynucleotides. The quality ofspecific PCR products obtained using synthetic primers will beindistinguishable from the product obtained from unamplified DNA. It isexpected that this approach will be useful for the mapping of genomes.

The polynucleotide to be shuffled can be produced as random ornon-random polynucleotides, at the discretion of the practitioner.

In Vivo Shuffling

In an embodiment of in vivo shuffling, the mixed population of thespecific nucleic acid sequence is introduced into bacterial oreukaryotic cells under conditions such that at least two differentnucleic acid sequences are present in each host cell. Thepolynucleotides can be introduced into the host cells by a variety ofdifferent methods. The host cells can be transformed with the smallerpolynucleotides using methods known in the art, for example treatmentwith calcium chloride. If the polynucleotides are inserted into a phagegenome, the host cell can be transfected with the recombinant phagegenome having the specific nucleic acid sequences. Alternatively, thenucleic acid sequences can be introduced into the host cell usingelectroporation, transfection, lipofection, biolistics, conjugation, andthe like.

In general, in this embodiment, the specific nucleic acids sequenceswill be present in vectors which are capable of stably replicating thesequence in the host cell. In addition, it is contemplated that thevectors will encode a marker gene such that host cells having the vectorcan be selected. This ensures that the mutated specific nucleic acidsequence can be recovered after introduction into the host cell.However, it is contemplated that the entire mixed population of thespecific nucleic acid sequences need not be present on a vectorsequence. Rather only a sufficient number of sequences need be clonedinto vectors to ensure that after introduction of the polynucleotidesinto the host cells each host cell contains one vector having at leastone specific nucleic acid sequence present therein. It is alsocontemplated that rather than having a subset of the population of thespecific nucleic acids sequences cloned into vectors, this subset may bealready stably integrated into the host cell.

It has been found that when two polynucleotides which have regions ofidentity are inserted into the host cells homologous recombinationoccurs between the two polynucleotides. Such recombination between thetwo mutated specific nucleic acid sequences will result in theproduction of double or triple hybrids in some situations.

It has also been found that the frequency of recombination is increasedif some of the mutated specific nucleic acid sequences are present onlinear nucleic acid molecules. Therefore, in a preferred embodiment,some of the specific nucleic acid sequences are present on linearpolynucleotides.

After transformation, the host cell transformants are placed underselection to identify those host cell transformants which containmutated specific nucleic acid sequences having the qualities desired.For example, if increased resistance to a particular drug is desiredthen the transformed host cells may be subjected to increasedconcentrations of the particular drug and those transformants producingmutated proteins able to confer increased drug resistance will beselected. If the enhanced ability of a particular protein to bind to areceptor is desired, then expression of the protein can be induced fromthe transformants and the resulting protein assayed in a ligand bindingassay by methods known in the art to identify that subset of the mutatedpopulation which shows enhanced binding to the ligand. Alternatively,the protein can be expressed in another system to ensure properprocessing.

Once a subset of the first recombined specific nucleic acid sequences(daughter sequences) having the desired characteristics are identified,they are then subject to a second round of recombination.

In the second cycle of recombination, the recombined specific nucleicacid sequences may be mixed with the original mutated specific nucleicacid sequences (parent sequences) and the cycle repeated as describedabove. In this way a set of second recombined specific nucleic acidssequences can be identified which have enhanced characteristics orencode for proteins having enhanced properties. This cycle can berepeated a number of times as desired.

It is also contemplated that in the second or subsequent recombinationcycle, a backcross can be performed. A molecular backcross can beperformed by mixing the desired specific nucleic acid sequences with alarge number of the wild-type sequence, such that at least one wild-typenucleic acid sequence and a mutated nucleic acid sequence are present inthe same host cell after transformation. Recombination with thewild-type specific nucleic acid sequence will eliminate those neutralmutations that may affect unselected characteristics such asimmunogenicity but not the selected characteristics.

In another embodiment of this invention, it is contemplated that duringthe first round a subset of the specific nucleic acid sequences can begenerated as smaller polynucleotides by slowing or halting their PCRamplification prior to introduction into the host cell. The size of thepolynucleotides must be large enough to contain some regions of identitywith the other sequences so as to homologously recombine with the othersequences. The size of the polynucleotides will range from 0.03 kb to100 kb more preferably from 0. 2 kb to 10 kb. It is also contemplatedthat in subsequent rounds, all of the specific nucleic acid sequencesother than the sequences selected from the previous round may beutilized to generate PCR polynucleotides prior to introduction into thehost cells.

The shorter polynucleotide sequences can be single-stranded ordouble-stranded. If the sequences were originally single-stranded andhave become double-stranded they can be denatured with heat, chemicalsor enzymes prior to insertion into the host cell. The reactionconditions suitable for separating the strands of nucleic acid are wellknown in the art.

The steps of this process can be repeated indefinitely, being limitedonly by the number of possible hybrids which can be achieved. After acertain number of cycles, all possible hybrids will have been achievedand further cycles are redundant.

In an embodiment the same mutated template nucleic acid is repeatedlyrecombined and the resulting recombinants selected for the desiredcharacteristic.

Therefore, the initial pool or population of mutated template nucleicacid is cloned into a vector capable of replicating in a bacteria suchas E. coli. The particular vector is not essential, so long as it iscapable of autonomous replication in E. coli. In a preferred embodiment,the vector is designed to allow the expression and production of anyprotein encoded by the mutated specific nucleic acid linked to thevector. It is also preferred that the vector contain a gene encoding fora selectable marker.

The population of vectors containing the pool of mutated nucleic acidsequences is introduced into the E. coli host cells. The vector nucleicacid sequences may be introduced by transformation, transfection orinfection in the case of phage. The concentration of vectors used totransform the bacteria is such that a number of vectors is introducedinto each cell. Once present in the cell, the efficiency of homologousrecombination is such that homologous recombination occurs between thevarious vectors. This results in the generation of hybrids (daughters)having a combination of mutations which differ from the original parentmutated sequences.

The host cells are then clonally replicated and selected for the markergene present on the vector. Only those cells having a plasmid will growunder the selection.

The host cells which contain a vector are then tested for the presenceof favorable mutations. Such testing may consist of placing the cellsunder selective pressure, for example, if the gene to be selected is animproved drug resistance gene. If the vector allows expression of theprotein encoded by the mutated nucleic acid sequence, then suchselection may include allowing expression of the protein so encoded,isolation of the protein and testing of the protein to determinewhether, for example, it binds with increased efficiency to the ligandof interest.

Once a particular daughter mutated nucleic acid sequence has beenidentified which confers the desired characteristics, the nucleic acidis isolated either already linked to the vector or separated from thevector. This nucleic acid is then mixed with the first or parentpopulation of nucleic acids and the cycle is repeated.

It has been shown that by this method nucleic acid sequences havingenhanced desired properties can be selected.

In an alternate embodiment, the first generation of hybrids are retainedin the cells and the parental mutated sequences are added again to thecells. Accordingly, the first cycle of Embodiment I is conducted asdescribed above. However, after the daughter nucleic acid sequences areidentified, the host cells containing these sequences are retained.

The parent mutated specific nucleic acid population, either aspolynucleotides or cloned into the same vector is introduced into thehost cells already containing the daughter nucleic acids. Recombinationis allowed to occur in the cells and the next generation ofrecombinants, or granddaughters are selected by the methods describedabove.

This cycle can be repeated a number of times until the nucleic acid orpeptide having the desired characteristics is obtained. It iscontemplated that in subsequent cycles, the population of mutatedsequences which are added to the preferred hybrids may come from theparental hybrids or any subsequent generation.

In an alternative embodiment, the invention provides a method ofconducting a “molecular” backcross of the obtained recombinant specificnucleic acid in order to eliminate any neutral mutations. Neutralmutations are those mutations which do not confer onto the nucleic acidor peptide the desired properties. Such mutations may however confer onthe nucleic acid or peptide undesirable characteristics. Accordingly, itis desirable to eliminate such neutral mutations. The method of thisinvention provide a means of doing so.

In this embodiment, after the hybrid nucleic acid, having the desiredcharacteristics, is obtained by the methods of the embodiments, thenucleic acid, the vector having the nucleic acid or the host cellcontaining the vector and nucleic acid is isolated.

The nucleic acid or vector is then introduced into the host cell with alarge excess of the wild-type nucleic acid. The nucleic acid of thehybrid and the nucleic acid of the wild-type sequence are allowed torecombine. The resulting recombinants are placed under the sameselection as the hybrid nucleic acid. Only those recombinants whichretained the desired characteristics will be selected. Any silentmutations which do not provide the desired characteristics will be lostthrough recombination with the wild-type DNA. This cycle can be repeateda number of times until all of the silent mutations are eliminated.

Thus the methods of this invention can be used in a molecular backcrossto eliminate unnecessary or silent mutations.

Utility

The in vivo recombination method of this invention can be performedblindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA and growth hormone. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

Scaffold-like regions separating regions of diversity in proteins may beparticularly suitable for the methods of this invention. The conservedscaffold determines the overall folding by self-association, whiledisplaying relatively unrestricted loops that mediate the specificbinding. Examples of such scaffolds are the immunoglobulin beta barrel,and the four-helix bundle. The methods of this invention can be used tocreate scaffold-like proteins with various combinations of mutatedsequences for binding.

The equivalents of some standard genetic matings may also be performedby the methods of this invention. For example, a “molecular” backcrosscan be performed by repeated mixing of the hybrid's nucleic acid withthe wild-type nucleic acid while selecting for the mutations ofinterest. As in traditional breeding, this approach can be used tocombine phenotypes from different sources into a background of choice.It is useful, for example, for the removal of neutral mutations thataffect unselected characteristics (ie. immunogenicity). Thus it can beuseful to determine which mutations in a protein are involved in theenhanced biological activity and which are not.

Peptide Display Methods The present method can be used to shuffle, by invitro and/or in vivo recombination by any of the disclosed methods, andin any combination, polynucleotide sequences selected by peptide displaymethods, wherein an associated polynucleotide encodes a displayedpeptide which is screened for a phenotype (eg., for affinity for apredetermined receptor (ligand).

An increasingly important aspect of bio-pharmaceutical drug developmentand molecular biology is the identification of peptide structures,including the primary amino acid sequences, of peptides orpeptidomimetics that interact with biological macromolecules, one methodof identifying peptides that possess a desired structure or functionalproperty, such as binding to a predetermined biological macromolecule(e.g., a receptor), involves the screening of a large library orpeptides for individual library members which possess the desiredstructure or functional property conferred by the amino acid sequence ofthe peptide.

In addition to direct chemical synthesis methods for generating peptidelibraries, several recombinant DNA methods also have been reported. Onetype involves the display of a peptide sequence, antibody, or otherprotein on the surface of a bacteriophage particle or cell. Generally,in these methods each bacteriophage particle or cell serves as anindividual library member displaying a single species of displayedpeptide in addition to the natural bacteriophage or cell proteinsequences. Each bacteriophage or cell contains the nucleotide sequenceinformation encoding the particular displayed peptide sequence; thus,the displayed peptide sequence can be ascertained by nucleotide sequencedetermination of an isolated library member.

A well-known peptide display method involves the presentation of apeptide sequence on the surface of a filamentous bacteriophage,typically as a fusion with a bacteriophage coat protein. Thebacteriophage library can be incubated with an immobilized,predetermined macromolecule or small molecule (e.g., a receptor) so thatbacteriophage particles which present a peptide sequence that binds tothe immobilized macromolecule can be differentially partitioned fromthose that do not present peptide sequences that bind to thepredetermined macromolecule. The bacteriophage particles (i.e., librarymembers) which are bound to the immobilized macromolecule are thenrecovered and replicated to amplify the selected bacteriophagesub-population for a subsequent round of affinity enrichment and phagereplication. After several rounds of affinity enrichment and phagereplication, the bacteriophage library members that are thus selectedare isolated and the nucleotide sequence encoding the displayed peptidesequence is determined, thereby identifying the sequence(s) of peptidesthat bind to the predetermined macromolecule (e.g., receptor). Suchmethods are further described in PCT patent publication Nos. 91/17271,91/18980, 91/19818 and 93/08278.

The latter PCT publication describes a recombinant DNA method for thedisplay of peptide ligands that involves the production of a library offusion proteins with each fusion protein composed of a first polypeptideportion, typically comprising a variable sequence, that is available forpotential binding to a predetermined macromolecule, and a secondpolypeptide portion that binds to DNA, such as the DNA vector encodingthe individual fusion protein. When transformed host cells are culturedunder conditions that allow for expression of the fusion protein, thefusion protein binds to the DNA vector encoding it. Upon lysis of thehost cell, the fusion protein/vector DNA complexes can be screenedagainst a predetermined macromolecule in much the same way asbacteriophage particles are screened in the phage-based display system,with the replication and sequencing of the DNA vectors in the selectedfusion protein/vector DNA complexes serving as the basis foridentification of the selected library peptide sequence(s).

Other systems for generating libraries of peptides and like polymershave aspects of both the recombinant and in vitro chemical synthesismethods. In these hybrid methods, cell-free enzymatic machinery isemployed to accomplish the in vitro synthesis of the library members(ie., peptides or polynucleotides). In one type of method, RNA moleculeswith the ability to bind a predetermined protein or a predetermined dyemolecule were selected by alternate rounds of selection and PCRamplification (Tuerk and Gold, 1990; Ellington and Szosta{dot over (k)},1990). A similar technique was used to identify DNA sequences which binda predetermined human transcription factor (Thiesen and Bach, 1990;Beaudry and Joyce, 1992; PCT patent publication Nos. 92/05258 and92/14843 *need inventor). In a similar fashion, the technique of invitro translation has been used to synthesize proteins of interest andhas been proposed as a method for generating large libraries ofpeptides. These methods which rely upon in vitro translation, generallycomprising stabilized polysome complexes, are described further in PCTpatent publication Nos. 88/08453, 90/05785, 90/07003, 91/02076,91/05058, and 92/02536. Applicants have described methods in whichlibrary members comprise a fusion protein having a first polypeptideportion with DNA binding activity and a second polypeptide portionhaving the library member unique peptide sequence; such methods aresuitable for use in cell-free in vitro selection formats, among others.

The displayed peptide sequences can be of varying lengths, typicallyfrom 3-5000 amino acids long or longer, frequently from 5-100 aminoacids long, and often from about 8-15 amino acids long. A library cancomprise library members having varying lengths of displayed peptidesequence, or may comprise library members having a fixed length ofdisplayed peptide sequence. Portions or all of the displayed peptidesequence(s) can be random, pseudorandom, defined set kernal, fixed, orthe like. The present display methods include methods for in vitro andin vivo display of single-chain antibodies, such as nascent scFv onpolysomes or scfv displayed on phage, which enable large-scale screeningof scfv libraries having broad diversity of variable region sequencesand binding specificities.

The present invention also provides random, pseudorandom, and definedsequence framework peptide libraries and methods for generating andscreening those libraries to identify useful compounds (e.g., peptides,including single-chain antibodies) that bind to receptor molecules orepitopes of interest or gene products that modify peptides or RNA in adesired fashion. The random, pseudorandom, and defined sequenceframework peptides are produced from libraries of peptide librarymembers that comprise displayed peptides or displayed single-chainantibodies attached to a polynucleotide template from which thedisplayed peptide was synthesized. The mode of attachment may varyaccording to the specific embodiment of the invention selected, and caninclude encapsulation in a phage particle or incorporation in a cell.

A method of affinity enrichment allows a very large library of peptidesand single-chain antibodies to be screened and the polynucleotidesequence encoding the desired peptide(s) or single-chain antibodies tobe selected. The polynucleotide can then be isolated and shuffled torecombine combinatorially the amino acid sequence of the selectedpeptide(s) (or predetermined portions thereof) or single-chainantibodies (or just VHI, VLI or CDR portions thereof). Using thesemethods, one can identify a peptide or single-chain antibody as having adesired binding affinity for a molecule and can exploit the process ofshuffling to converge rapidly to a desired high-affinity peptide orscfv. The peptide or antibody can then be synthesized in bulk byconventional means for any suitable use (e.g., as a therapeutic ordiagnostic agent).

A significant advantage of the present invention is that no priorinformation regarding an expected ligand structure is required toisolate peptide ligands or antibodies of interest. The peptideidentified can have biological activity, which is meant to include atleast specific binding affinity for a selected receptor molecule and, insome instances, will further include the ability to block the binding ofother compounds, to stimulate or inhibit metabolic pathways, to act as asignal or messenger, to stimulate or inhibit cellular activity, and thelike.

The present invention also provides a method for shuffling a pool ofpolynucleotide sequences selected by affinity screening a library ofpolysomes displaying nascent peptides (including single-chainantibodies) for library members which bind to a predetermined receptor(e.g., a mammalian proteinaceous receptor such as, for example, apeptidergic hormone receptor, a cell surface receptor, an intracellularprotein which binds to other protein(s) to form intracellular proteincomplexes such as hetero-dimers and the like) or epitope (e.g., animmobilized protein, glycoprotein, oligosaccharide, and the like).

Polynucleotide sequences selected in a first selection round (typicallyby affinity selection for binding to a receptor (e.g., a ligand)).by anyof these methods are pooled and the pool(s) is/are shuffled by in vitroand/or in vivo recombination to produce a shuffled pool comprising apopulation of recombined selected polynucleotide sequences. Therecombined selected polynucleotide sequences are subjected to at leastone subsequent selection round. The polynucleotide sequences selected inthe subsequent selection round(s) can be used directly, sequenced,and/or subjected to one or more additional rounds of shuffling andsubsequent selection. Selected sequences can also be back-crossed withpolynucleotide sequences encoding neutral sequences (i.e., havinginsubstantial functional effect on binding), such as for example byback-crossing with a wild-type or naturally-occurring sequencesubstantially identical to a selected sequence to produce native-likefunctional peptides, which may be less immunogenic. Generally, duringback-crossing subsequent selection is applied to retain the property ofbinding to the predetermined receptor (ligand).

Prior to or concomitant with the shuffling of selected sequences, thesequences can be mutagenized. In one embodiment, selected librarymembers are cloned in a prokaryotic vector (e.g., plasmid, phagemid, orbacteriophage) wherein a collection of individual. colonies (or plaques)representing discrete library members are produced. Individual selectedlibrary members can then be manipulated (e.g., by site-directedmutagenesis, cassette mutagenesis, chemical mutagenesis, PCRmutagenesis, and the like) to generate a collection of library membersrepresenting a kernal of sequence diversity based on the sequence of theselected library member. The sequence of an individual selected librarymember or pool can be manipulated to incorporate random mutation,pseudorandom mutation, defined kernal mutation (i e., comprising variantand invariant residue positions and/or comprising variant residuepositions which can comprise a residue selected from a defined subset ofamino acid residues), codon-based mutation, and the like, eithersegmentally or over the entire length of the individual selected librarymember sequence. The mutagenized selected library members are thenshuffled by in vitro and/or in vivo recombinatorial shuffling asdisclosed herein.

The invention also provides peptide libraries comprising a plurality ofindividual library members of the invention, wherein (1) each individuallibrary member of said plurality comprises a sequence produced byshuffling of a pool of selected sequences, and (2) each individuallibrary member comprises a variable peptide segment sequence orsingle-chain antibody segment sequence which is distinct from thevariable peptide segment sequences or single-chain antibody sequences ofother individual library members in said plurality (although somelibrary members may be present in more than one copy per library due touneven amplification, stochastic probability or the like).

The invention also provides a product-by-process, wherein selectedpolynucleotide sequences having (or encoding a peptide having) apredetermined binding specificity are formed by the process of: (1)screening a displayed peptide or displayed single-chain antibody libraryagainst a predetermined receptor (e.g., ligand) or epitope (e.g.,antigen macromolecule) and identifying and/or enriching library memberswhich bind to the predetermined receptor or epitope to produce a pool ofselected library members, (2) shuffling by recombination the selectedlibrary members (or amplified or cloned copies thereof) which binds thepredetermined epitope and has been thereby isolated and/or enriched fromthe library to generate a shuffled library, and (3) screening theshuffled library against the predetermined receptor (e.g., ligand) orepitope (e.g., antigen macromolecule) and identifying and/or enrichingshuffled library members which bind to the predetermined receptor orepitope to produce a pool of selected shuffled library members.

Antibody Display and Screening Methods

The present method can be used to shuffle, by in vitro and/or in vivorecombination by any of the disclosed methods, and in any combination,polynucleotide sequences selected by antibody display methods, whereinan associated polynucleotide encodes a displayed antibody which isscreened for a phenotype (e.g., for affinity for binding a predeterminedantigen (ligand).

Various molecular genetic approaches have been devised to capture thevast immunological repertoire represented by the extremely large numberof distinct variable regions which can be present in immunoglobulinchains. The naturally-occurring germ line immunoglobulin heavy chainlocus is composed of separate tandem arrays of variable segment geneslocated upstream of a tandem array of diversity segment genes, which arethemselves located upstream of a tandem array of joining (i) regiongenes, which are located upstream of the constant region genes. During Blymphocyte development, V-D-J rearrangement occurs wherein a heavy chainvariable region gene (VH) is formed by rearrangement to form a fused Dsegment followed by rearrangement with a V segment to form a V-D-Jjoined product gene which, if productively rearranged, encodes afunctional variable region (VH) of a heavy chain. Similarly, light chainloci rearrange one of several V segments with one of several J segmentsto form a gene encoding the variable region (VL) of a light chain.

The vast repertoire of variable regions possible in immunoglobulinsderives in part from the numerous combinatorial possibilities of joiningV and i segments (and, in the case of heavy chain loci, D segments)during rearrangement in B cell development. Additional sequencediversity in the heavy chain variable regions arises from non-uniformrearrangements of the D segments during V-D-J joining and from N regionaddition. Further, antigen-selection of specific B cell clones selectsfor higher affinity variants having non-germline mutations in one orboth of the heavy and light chain variable regions; a phenomenonreferred to as “affinity maturation” or “affinity sharpening”.Typically, these “affinity sharpening” mutations cluster in specificareas of the variable region, most commonly in thecomplementarity-determining regions (CDRs).

In order to overcome many of the limitations in producing andidentifying high-affinity immunoglobulins through antigen-stimulated βcell development (i.e., immunization), various prokaryotic expressionsystems have been developed that can be manipulated to producecombinatorial antibody libraries which may be screened for high-affinityantibodies to specific antigens. Recent advances in the expression ofantibodies in Escherichia coli and bacteriophage systems (see,*“Alternative Peptide Display Methods”, infra) have raised thepossibility that virtually any specificity can be obtained by eithercloning antibody genes from characterized hybridomas or by de novoselection using antibody gene libraries (e.g., from Ig CDNA).

Combinatorial libraries of antibodies have been generated inbacteriophage lambda expression systems which may be screened asbacteriophage plaques or as colonies of lysogens (Huse et al,. 1989);Caton and Koprowski, 1990; Mullinax et al., 1990; Persson et al., 1991).Various embodiments of bacteriophage antibody display libraries andlambda phage expression libraries have been described (Kang et al.,1991; Clackson et al., 1991; McCafferty et al., 1990; Burton et al.,1991; Hoogenboom et al., 1991; Chang et al., 1991; Breitling et al.,1991; Marks et al., 1991, p.581; Barbas et al., 1992; Hawkins andWinter, 1992; Marks et al., 1992, p. 779; Marks et al., 1992, p. 16007;Lowman et al., 1991; Lerner et al., 1992, incorporated herein byreference). Typically, a bacteriophage antibody display library isscreened with a receptor (e.g., polypeptide, carbohydrate, glycoprotein,nucleic acid) that is immobilized (e.g., by covalent linkage to achromatography resin to enrich for reactive phage by affinitychromatography) and/or labeled (e.g., to screen plaque or colony lifts).

One particularly advantageous approach has been the use of so-calledsingle-chain fragment variable (scfv) libraries (Marks et al., supra, p.779; Winter and Milstein, 1991; Clackson et al., supra; Marks et al.,supra, p. 581; Chaudhary et al., 1990; Chiswell et al., 1992; McCaffertyet al., supra; and Huston et al., 1988). Various embodiments of scfvlibraries displayed on bacteriophage coat proteins have been described.

Beginning in 1988, single-chain analogues of Fv fragments and theirfusion proteins have been reliably generated by antibody engineeringmethods. The first step generally involves obtaining the genes encodingVH and VL domains with desired binding properties; these V genes may beisolated from a specific hybridoma cell line, selected from acombinatorial V-gene library, or made by V gene synthesis. Thesingle-chain Fv is formed by connecting the component V genes with anoligonucleotide that encodes an appropriately designed linker peptide,such as (Gly-Gly-Gly-Gly-Ser)3 or equivalent linker peptide(s). Thelinker bridges the C-terminus of the first V region and N-terminus ofthe second, ordered as either VH-linker-VL or VL-linker-VH′ Inprinciple, the scfv binding site can faithfully replicate both theaffinity and specificity of its parent antibody combining site.

Thus, scfv fragments are comprised of VH and VL domains linked into asingle polypeptide chain by a flexible linker peptide. After the scfvgenes are assembled, they are cloned into a phagemid and expressed atthe tip of the M13 phage (or similar filamentous bacteriophage) asfusion proteins with the bacteriophage PIII (gene 3) coat protein.Enriching for phage expressing an antibody of interest is accomplishedby panning the recombinant phage displaying a population scfv forbinding to a predetermined epitope (e.g., target antigen, receptor).

The linked polynucleotide of a library member provides the basis forreplication of the library member after a screening or selectionprocedure, and also provides the basis for the determination, bynucleotide sequencing, of the identity of the displayed peptide sequenceor VH and VL amino acid sequence. The displayed peptide (s) orsingle-chain antibody (e. g., scfv) and/or its VH and VL domains ortheir CDRs can be cloned and expressed in a suitable expression system.Often polynucleotides encoding the isolated VH and VL domains will beligated to polynucleotides encoding constant regions (CH and CL) to formpolynucleotides encoding complete antibodies (e.g., chimeric orfully-human), antibody fragments, and the like. Often polynucleotidesencoding the isolated CDRs will be grafted into polynucleotides encodinga suitable variable region framework (and optionally constant regions)to form polynucleotides encoding complete antibodies (e.g., humanized orfully-human), antibody fragments, and the like. Antibodies can be usedto isolate preparative quantities of the antigen by immunoaffinitychromatography. Various other uses of such antibodies are to diagnoseand/or stage disease (e.g., neoplasia) and for therapeutic applicationto treat disease, such as for example: neoplasia, autoimmune disease,AIDS, cardiovascular disease, infections, and the like.

Various methods have been reported for increasing the combinatorialdiversity of a scfv library to broaden the repertoire of binding species(idiotype spectrum) The use of PCR has permitted the variable regions tobe rapidly cloned either from a specific hybridoma source or as a genelibrary from non-immunized cells, affording combinatorial diversity inthe assortment of VH and VL cassettes which can be combined.Furthermore, the VH and VL cassettes can themselves be diversified, suchas by random, pseudorandom, or directed mutagenesis. Typically, VH andVL cassettes are diversified in or near the complementarity-determiningregions (CDRS), often the third CDR, CDR3. Enzymatic inverse PCRmutagenesis has been shown to be a simple and reliable method forconstructing relatively large libraries of scfv site-directed hybrids(Stemmer el al., 1993), as has error-prone PCR and chemical mutagenesis(Deng et al., 1994). Riechmann et al. (1993) Biochemistry 32:8848 showedsemi-rational design of an antibody scfv fragment using site-directedrandomization by degenerate oligonucleotide PCR and subsequent phagedisplay of the resultant scfv hybrids. * Barbas et al. (1992) on.cit.attempted to circumvent the problem of limited repertoire sizesresulting from using biased variable region sequences by randomizing thesequence in a synthetic CDR region of a human tetanus toxoid-bindingFab.

CDR randomization has the potential to create approximately 1×10²⁰ CDRsfor the heavy chain CDR3 alone, and a roughly similar number of variantsof the heavy chain CDR1 and CDR2, and light chain CDR1-3 variants. Takenindividually or together, the combination possibilities of CDRrandomization of heavy and/or light chains requires generating aprohibitive number of bacteriophage clones to produce a clone libraryrepresenting all possible combinations, the vast majority of which willbe non-binding. Generation of such large numbers of primarytransformants is not feasible with current transformation technology andbacteriophage display systems. For example, Barbas et al. (1992) op.cit.only generated 5×10⁷ transformants, which represents only a tinyfraction of the potential diversity of a library of thoroughlyrandomized CDRS.

Despite these substantial limitations, bacteriophage display of scfvhave already yielded a variety of useful antibodies and antibody fusionproteins. A bispecific single chain antibody has been shown to mediateefficient tumor cell lysis (Gruber et al., 1994). Intracellularexpression of an anti-Rev scfv has been shown to inhibit HIV-1 virusreplication in vitro (Duan et al., 1994), and intracellular expressionof an anti-p2lrar, scfv has been shown to inhibit meiotic maturation ofXenopus oocytes (Biocca et al., 1993). Recombinant scfv which can beused to diagnose HIV infection have also been reported, demonstratingthe diagnostic utility of scfv (Lilley et al., 1994). Fusion proteinswherein an scFv is linked to a second polypeptide, such as a toxin orfibrinolytic activator protein, have also been reported (Holvost et al.,1992; Nicholls et al., 1993).

If it were possible to generate scfv libraries having broader antibodydiversity and overcoming many of the limitations of conventional CDRmutagenesis and randomization methods which can cover only a very tinyfraction of the potential sequence combinations, the number and qualityof scfv antibodies suitable for therapeutic and diagnostic use could bevastly improved. To address this, the in vitro and in vivo shufflingmethods of the invention are used to recombine CDRs which have beenobtained (typically via PCR amplification or cloning) from nucleic acidsobtained from selected displayed antibodies. Such displayed antibodiescan be displayed on cells, on bacteriophage particles, on polysomes, orany suitable antibody display system wherein the antibody is associatedwith its encoding nucleic acid(s). In a variation, the CDRs areinitially obtained from MRNA (or CDNA) from antibody-producing cells(e.g., plasma cells/splenocytes from an immunized wild-type mouse, ahuman, or a transgenic mouse capable of making a human antibody as inW092/03918, W093/12227, and W094/25585), including hybridomas derivedtherefrom.

Polynucleotide sequences selected in a first selection round (typicallyby affinity selection for displayed antibody binding to an antigen(e.g., a ligand) by any of these methods are pooled and the pool(s)is/are shuffled by in vitro and/or in vivo recombination, especiallyshuffling of CDRs (typically shuffling heavy chain CDRs with other heavychain CDRs and light chain CDRs with other light chain CDRS) to producea shuffled pool comprising a population of recombined selectedpolynucleotide sequences. The recombined selected polynucleotidesequences are expressed in a selection format as a displayed antibodyand subjected to at least one subsequent selection round. Thepolynucleotide sequences selected in the subsequent selection round(s)can be used directly, sequenced, and/or subjected to one or moreadditional rounds of shuffling and subsequent selection until anantibody of the desired binding affinity is obtained. Selected sequencescan also be back-crossed with polynucleotide sequences encoding neutralantibody framework sequences (i.e., having insubstantial functionaleffect on antigen binding), such as for example by back-crossing with ahuman variable region framework to produce human-like sequenceantibodies. Generally, during back-crossing subsequent selection isapplied to retain the property of binding to the predetermined antigen.

Alternatively, or in combination with the noted variations, the valencyof the target epitope may be varied to control the average bindingaffinity of selected scfv library members. The target epitope can bebound to a surface or substrate at varying densities, such as byincluding a competitor epitope, by dilution, or by other method known tothose in the art. A high density (valency) of predetermined epitope canbe used to enrich for scfv library members which have relatively lowaffinity, whereas a low density (valency) can preferentially enrich forhigher affinity scfv library members.

For generating diverse variable segments, a collection of syntheticoligonucleotides encoding random, pseudorandom, or a defined sequencekernal set of peptide sequences can be inserted by ligation into apredetermined site (e.g., a CDR). Similarly, the sequence diversity ofone or more CDRs of the single-chain antibody cassette(s) can beexpanded by mutating the CDR(s) with site-directed mutagenesis,CDR-replacement, and the like. The resultant DNA molecules can bepropagated in a host for cloning and amplification prior to shuffling,or can be used directly (i.e., may avoid loss of diversity which mayoccur upon propagation in a host cell) and the selected library memberssubsequently shuffled.

Displayed peptide/polynucleotide complexes (library members) whichencode a variable segment peptide sequence of interest or a single-chainantibody of interest are selected from the library by an affinityenrichment technique. This is accomplished by means of a immobilizedmacromolecule or epitope specific for the peptide sequence of interest,such as a receptor, other macromolecule, or other epitope species.Repeating the affinity selection procedure provides an enrichment oflibrary members encoding the desired sequences, which may then beisolated for pooling and shuffling, for sequencing, and/or for furtherpropagation and affinity enrichment.

The library members without the desired specificity are removed bywashing. The degree and stringency of washing required will bedetermined for each peptide sequence or single-chain antibody ofinterest and the immobilized predetermined macromolecule or epitope. Acertain degree of control can be exerted over the bindingcharacteristics of the nascent peptide/DNA complexes recovered byadjusting the conditions of the binding incubation and the subsequentwashing. The temperature, pH, ionic strength, divalent cationsconcentration, and the volume and duration of the washing will selectfor nascent peptide/DNA complexes within particular ranges of affinityfor the immobilized macromolecule. Selection based on slow dissociationrate, which is usually predictive of high affinity, is often the mostpractical route. This may be done either by continued incubation in thepresence of a saturating amount of free predetermined macromolecule, orby increasing the volume, number, and length of the washes. In eachcase, the rebinding of dissociated nascent peptide/DNA or peptide/RNAcomplex is prevented, and with increasing time, nascent peptide/DNA orpeptide/RNA complexes of higher and higher affinity are recovered.

Additional modifications of the binding and washing procedures may beapplied to find peptides with special characteristics. The affinities ofsome peptides are dependent on ionic strength or cation concentration.This is a useful characteristic for peptides that will be used inaffinity purification of various proteins when gentle conditions forremoving the protein from the peptides are required.

One variation involves the use of multiple binding targets (multipleepitope species, multiple receptor species), such that a scfv librarycan be simultaneously screened for a multiplicity of scfv which havedifferent binding specificities. Given that the size of a scfv libraryoften limits the diversity of potential scfv sequences, it is typicallydesirable to us scfv libraries of as large a size as possible. The timeand economic considerations of generating a number of very largepolysome scFv-display libraries can become prohibitive. To avoid thissubstantial problem, multiple predetermined epitope species (receptorspecies) can be concomitantly screened in a single library, orsequential screening against a number of epitope species can be used. Inone variation, multiple target epitope species, each encoded on aseparate bead (or subset of beads), can be mixed and incubated with apolysome-display scfv library under suitable binding conditions. Thecollection of beads, comprising multiple epitope species, can then beused to isolate, by affinity selection, scfv library members. Generally,subsequent affinity screening rounds can include the same mixture ofbeads, subsets thereof, or beads containing only one or two individualepitope species. This approach affords efficient screening, and iscompatible with laboratory automation, batch processing, and highthroughput screening methods.

A variety of techniques can be used in the present invention todiversify a peptide library or single-chain antibody library, or todiversify, prior to or concomitant with shuffling, around variablesegment peptides found in early rounds of panning to have sufficientbinding activity to the predetermined macromolecule or epitope. In oneapproach, the positive selected peptide/polynucleotide complexes (thoseidentified in an early round of affinity enrichment) are sequenced todetermine the identity of the active peptides. Oligonucleotides are thensynthesized based on these active peptide sequences, employing a lowlevel of all bases incorporated at each step to produce slightvariations of the primary oligonucleotide sequences. This mixture of(slightly) degenerate oligonucleotides is then cloned into the variablesegment sequences at the appropriate locations. This method producessystematic, controlled variations of the starting peptide sequences,which can then be shuffled. It requires, however, that individualpositive nascent peptide/polynucleotide complexes be sequenced beforemutagenesis, and thus is useful for expanding the diversity of smallnumbers of recovered complexes and selecting variants having higherbinding affinity and/or higher binding specificity. In a variation,mutagenic PCR amplification of positive selected peptide/polynucleotidecomplexes (especially of the variable region sequences, theamplification products of which are shuffled in vitro and/or in vivo andone or more additional rounds of screening is done prior to sequencing.The same general approach can be employed with single-chain antibodiesin order to expand the diversity and enhance the bindingaffinity/specificity, typically by diversifying CDRs or adjacentframework regions prior to or concomitant with shuffling. If desired,shuffling reactions can be spiked with mutagenic oligonucleotidescapable of in vitro recombination with the selected library members canbe included. Thus, mixtures of synthetic oligonucleotides and PCRproduced polynucleotides (synthesized by error-prone or high-fidelitymethods) can be added to the in vitro shuffling mix and be incorporatedinto resulting shuffled library members (shufflants).

The present invention of shuffling enables the generation of a vastlibrary of CDR-variant single-chain antibodies. One way to generate suchantibodies is to insert synthetic CDRs into the single-chain antibodyand/or CDR randomization prior to or concomitant with shuffling. Thesequences of the synthetic CDR cassettes are selected by referring toknown sequence data of human CDR and are selected in the discretion ofthe practitioner according to the following guidelines: synthetic CDRswill have at least 40 percent positional sequence identity to known CDRsequences, and preferably will have at least 50 to 70 percent positionalsequence identity to known CDR sequences. For example, a collection ofsynthetic CDR sequences can be generated by synthesizing a collection ofoligonucleotide sequences on the basis of naturally-occurring human CDRsequences listed in Kabat et al. (1991) op.cit.; the pool (s) ofsynthetic CDR sequences are calculated to encode CDR peptide sequenceshaving at least 40 percent sequence identity to at least one knownnaturally-occurring human CDR sequence. Alternatively, a collection ofnaturally-occurring CDR sequences may be compared to generate consensussequences so that amino acids used at a residue position frequently(i.e., in at least 5 percent of known CDR sequences) are incorporatedinto the synthetic CDRs at the corresponding position(s). Typically,several (e.g., 3 to about 50) known CDR sequences are compared andobserved natural sequence variations between the known CDRs aretabulated, and a collection of oligonucleotides encoding CDR peptidesequences encompassing all or most permutations of the observed naturalsequence variations is synthesized. For example but not for limitation,if a collection of human VH CDR sequences have carboxy-terminal aminoacids which are either Tyr, Val, Phe, or Asp, then the pool(s) ofsynthetic CDR oligonucleotide sequences are designed to allow thecarboxy-terminal CDR residue to be any of these amino acids. In someembodiments, residues other than those which naturally-occur at aresidue position in the collection of CDR sequences are incorporated:conservative amino acid substitutions are frequently incorporated and upto 5 residue positions may be varied to incorporate non-conservativeamino acid substitutions as compared to known naturally-occurring CDRsequences. Such CDR sequences can be used in primary library members(prior to first round screening) and/or can be used to spike in vitroshuffling reactions of selected library member sequences. Constructionof such pools of defined and/or degenerate sequences will be readilyaccomplished by those of ordinary skill in the art.

The collection of synthetic CDR sequences comprises at least one memberthat is not known to be a naturally-occurring CDR sequence. It is withinthe discretion of the practitioner to include or not include a portionof random or pseudorandom sequence corresponding to N region addition inthe heavy chain CDR; the N region sequence ranges from 1 nucleotide toabout 4 nucleotides occurring at V-D and D-J junctions. A collection ofsynthetic heavy chain CDR sequences comprises at least about 100 uniqueCDR sequences, typically at least about 1,000 unique CDR sequences,preferably at least about 10,000 unique CDR sequences, frequently morethan 50,000 unique CDR sequences; however, usually not more than about1×10 6 unique CDR sequences are included in the collection, althoughoccasionally 1×107 to 1×108 unique CDR sequences are present, especiallyif conservative amino acid substitutions are permitted at positionswhere the conservative amino acid substituent is not present or is rare(i.e., less than 0.1 percent) in that position in naturally-occurringhuman CDRS. In general, the number of unique CDR sequences included in alibrary should not exceed the expected number of primary transformantsin the library by more than a factor of 10. Such single-chain antibodiesgenerally bind of about at least 1×10 m-, preferably with an affinity ofabout at least 5×10⁷ M-1, more preferably with an affinity of at least1×10⁸ M-1 to 1×10⁹ M-1 or more, sometimes up to 1×10¹⁰ M-1 or more.Frequently, the predetermined antigen is a human protein, such as forexample a human cell surface antigen (e. g., CD4, CD8, IL-2 receptor,EGF receptor, PDGF receptor), other human biological macromolecule(e.g., thrombomodulin, protein C, carbohydrate antigen, sialyl Lewisantigen, Lselectin), or nonhuman disease associated macromolecule (e.g.,bacterial LPS, virion capsid protein or envelope glycoprotein) and thelike.

High affinity single-chain antibodies of the desired specificity can beengineered and expressed in a variety of systems. For example, scfv havebeen produced in plants (Firek et al., 1993) and can be readily made inprokaryotic systems (Owens and Young, 1994; Johnson and Bird, 1991).Furthermore, the single-chain antibodies can be used as a basis forconstructing whole antibodies or various fragments thereof(Kettleborough et al., 1994). The variable region encoding sequence maybe isolated (e.g., by PCR amplification or subcloning) and spliced to asequence encoding a desired human constant region to encode a humansequence antibody more suitable for human therapeutic uses whereimmunogenicity is preferably minimized. The polynucleotide(s) having theresultant fully human encoding sequence(s) can be expressed in a hostcell (e.g., from an expression vector in a mammalian cell) and purifiedfor pharmaceutical formulation.

The DNA expression constructs will typically include an expressioncontrol DNA sequence operably linked to the coding sequences, includingnaturally-associated or heterologous promoter regions. Preferably, theexpression control sequences will be eukaryotic promoter systems invectors capable of transforming or transfecting eukaryotic host cells.Once the vector has been incorporated into the appropriate host, thehost is maintained under conditions suitable for high level expressionof the nucleotide sequences, and the collection and purification of themutant’ “engineered” antibodies.

As stated previously, the DNA sequences will be expressed in hosts afterthe sequences have been operably linked to an expression controlsequence (i.e., positioned to ensure the transcription and translationof the structural gene). These expression vectors are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosomal DNA. Commonly, expression vectors willcontain selection markers, e.g., tetracycline or neomycin, to permitdetection of those cells transformed with the desired DNA sequences(see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein byreference).

In addition to eukaryotic microorganisms such as yeast, mammalian tissuecell culture may also be used to produce the polypeptides of the presentinvention (see, Winnacker, 1987), which is incorporated herein byreference). Eukaryotic cells are actually preferred, because a number ofsuitable host cell lines capable of secreting intact immunoglobulinshave been developed in the art, and include the CHO cell lines, variousCOS cell lines, HeLa cells, and myeloma cell lines, but preferablytransformed Bcells or hybridomas. Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter, an enhancer (Queen et al., 1986), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, cytomegalovirus, SV40, Adenovirus, BovinePapilloma Virus, and the like.

Eukaryotic DNA transcription can be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting sequences of between10 to 300 bp that increase transcription by a promoter. Enhancers caneffectively increase transcription when either 51 or 31 to thetranscription unit. They are also effective if located within an intronor within the coding sequence itself. Typically, viral enhancers areused, including SV40 enhancers, cytomegalovirus enhancers, polyomaenhancers, and adenovirus enhancers. Enhancer sequences from mammaliansystems are also commonly used, such as the mouse immunoglobulin heavychain enhancer.

Mammalian expression vector systems will also typically include aselectable marker gene. Examples of suitable markers include, thedihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), orprokaryotic genes conferring drug resistance. The first two marker genesprefer the use of mutant cell lines that lack the ability to growwithout the addition of thymidine to the growth medium. Transformedcells can then be identified by their ability to grow onnon-supplemented media. Examples of prokaryotic drug resistance genesuseful as markers include genes conferring resistance to G418,mycophenolic acid and hygromycin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment.lipofection, or electroporation may be used for other cellular hosts.Other methods used to transform mammalian cells include the use ofPolybrene, protoplast fusion, liposomes, electroporation, andmicro-injection (see, generally, Sambrook et al., supra).

Once expressed, the antibodies, individual mutated immunoglobulinchains, mutated antibody fragments, and other immunoglobulinpolypeptides of the invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,fraction column chromatography, gel electrophoresis and the like (see,generally, Scopes, 1982). Once purified, partially or to homogeneity asdesired, the polypeptides may then be used therapeutically or indeveloping and performing assay procedures, immunofluorescent stainings,and the like (see, generally, Lefkovits and Pernis, 1979 and 1981).

The antibodies generated by the method of the present invention can beused for diagnosis and therapy. By way of illustration and notlimitation, they can be used to treat cancer, autoimmune diseases, orviral infections. For treatment of cancer, the antibodies will typicallybind to an antigen expressed preferentially on cancer cells, such aserbB-2, CEA, CD33, and many other antigens and binding members wellknown to those skilled in the art.

Two-Hybrid Based Screening Assays

Shuffling can also be used to recombinatorially diversify a pool ofselected library members obtained by screening a two-hybrid screeningsystem to identify library members which bind a predeterminedpolypeptide sequence. The selected library members are pooled andshuffled by in vitro and/or in vivo recombination. The shuffled pool canthen be screened in a yeast two hybrid system to select library memberswhich bind said predetermined polypeptide sequence (e. g., and SH2domain) or which bind an alternate predetermined polypeptide sequence(e.g., an SH2 domain from another protein species).

An approach to identifying polypeptide sequences which bind to apredetermined polypeptide sequence has been to use a so-called“two-hybrid” system wherein the predetermined polypeptide sequence ispresent in a fusion protein (Chien et al., 1991). This approachidentifies protein-protein interactions in vivo through reconstitutionof a transcriptional activator (Fields and Song, 1989), the yeast Gal4transcription protein. Typically, the method is based on the propertiesof the yeast Gal4 protein, which consists of separable domainsresponsible for DNA-binding and transcriptional activation.Polynucleotides encoding two hybrid proteins, one consisting of theyeast Gal4 DNA-binding domain fused to a polypeptide sequence of a knownprotein and the other consisting of the Gal4 activation domain fused toa polypeptide sequence of a second protein, are constructed andintroduced into a yeast host cell. Intermolecular binding between thetwo fusion proteins reconstitutes the Gal4 DNA-binding domain with theGal4 activation domain, which leads to the transcriptional activation ofa reporter gene (e.g., lacz, HIS3) which is operably linked to a Gal4binding site. Typically, the two-hybrid method is used to identify novelpolypeptide sequences which interact with a known protein (Silver andHunt, 1993; Durfee et al., 1993; Yang et al., 1992; Luban et al., 1993;Hardy et al., 1992; Bartel et al., 1993; and Vojtek et al., 1993).However, variations of the two-hybrid method have been used to identifymutations of a known protein that affect its binding to a second knownprotein (Li and Fields, 1993; Lalo et al., 1993; Jackson et al., 1993;and Madura et al., 1993). Two-hybrid systems have also been used toidentify interacting structural domains of two known proteins (Bardwellet al., 1993; Chakrabarty et al., 1992; Staudinger et al., 1993; andMilne and Weaver 1993) or domains responsible for oligomerization of asingle protein (Iwabuchi et al., 1993; Bogerd et al., 1993). Variationsof two-hybrid systems have been used to study the in vivo activity of aproteolytic enzyme (Dasmahapatra et al., 1992). Alternatively, an E.coli/BCCP interactive screening system (Germino et al., 1993; Guarente,1993) can be used to identify interacting protein sequences (i.e.,protein sequences which heterodimerize or form higher orderheteromultimers). Sequences selected by a two-hybrid system can bepooled and shuffled and introduced into a two-hybrid system for one ormore subsequent rounds of screening to identify polypeptide sequenceswhich bind to the hybrid containing the predetermined binding sequence.The sequences thus identified can be compared to identify consensussequence(s) and consensus sequence kernals.

In general, standard techniques of recombination DNA technology aredescribed in various publications. e.g. Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory; Ausubel etal., 1987, Current Protocols in Molecular Biology, vols. 1 and 2 andsupplements, and Berger and Kimmel, Methods in Enzymology, Volume 152,Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., SanDiego, Calif., each of which is incorporated herein in their entirety byreference. Polynucleotide modifying enzymes were used according to themanufacturer's recommendations. Oligonucleotides were synthesized on anApplied Biosystems Inc. Model 394 DNA synthesizer using ABI chemicals.If desired, PCR amplimers for amplifying a predetermined DNA sequencemay be selected at the discretion of the practitioner.

One microgram samples of template DNA are obtained and treated with U.V.light to cause the formation of dimers, including TT dimers,particularly purine dimers. U.V. exposure is limited so that only a fewphotoproducts are generated per gene on the template DNA sample.Multiple samples are treated with U.V. light for varying periods of timeto obtain template DNA samples with varying numbers of dimers from U.V.exposure.

A random priming kit which utilizes a non-proofreading polymease (forexample, Prime-It II Random Primer Labeling kit by Stratagene CloningSystems) is utilized to generate different size polynucleotides bypriming at random sites on templates which are prepared by U.V. light(as described above) and extending along the templates. The primingprotocols such as described in the Prime-It II Random Primer Labelingkit may be utilized to extend the primers. The dimers formed by U.V.exposure serve as a roadblock for the extension by the non-proofreadingpolymerase. Thus, a pool of random size polynucleotides is present afterextension with the random primers is finished.

The present invention is further directed to a method for generating aselected mutant polynucleotide sequence (or a population of selectedpolynucleotide sequences) typically in the form of amplified and/orcloned polynucleotides, whereby the selected polynucleotide sequences(s)possess at least one desired phenotypic characteristic (e.g., encodes apolypeptide, promotes transcription of linked polynucleotides, binds aprotein, and the like) which can be selected for. One method foridentifying hybrid polypeptides that possess a desired structure orfunctional property, such as binding to a predetermined biologicalmacromolecule (e.g., a receptor), involves the screening of a largelibrary of polypeptides for individual library members which possess thedesired structure or functional property conferred by the amino acidsequence of the polypeptide.

In one embodiment, the present invention provides a method forgenerating libraries of displayed polypeptides or displayed antibodiessuitable for affinity interaction screening or phenotypic screening. Themethod comprises (1) obtaining a first plurality of selected librarymembers comprising a displayed polypeptide or displayed antibody and anassociated polynucleotide encoding said displayed polypeptide ordisplayed antibody, and obtaining said associated polynucleotides orcopies thereof wherein said associated polynucleotides comprise a regionof substantially identical sequences, optimally introducing mutationsinto said polynucleotides or copies, (2) pooling the polynucleotides orcopies, (3) producing smaller or shorter polynucleotides by interruptinga random or particularized priming and synthesis process or anamplification process, and (4) performing amplification, preferably PCRamplification, and optionally mutagenesis to homologously recombine thenewly synthesized polynucleotides.

It is a particularly preferred object of the invention to provide aprocess for producing hybrid polynucleotides which express a usefulhybrid polypeptide by a series of steps comprising:

-   -   (a) producing polynucleotides by interrupting a polynucleotide        amplification or synthesis process with a means for blocking or        interrupting the amplification or synthesis process and thus        providing a plurality of smaller or shorter polynucleotides due        to the replication of the polynucleotide being in various stages        of completion;    -   (b) adding to the resultant population of single- or        double-stranded polynucleotides one or more single- or        double-stranded oligonucleotides, wherein said added        oligonucleotides comprise an area of identity in an area of        heterology to one or more of the single- or double-stranded        polynucleotides of the population;    -   (c) denaturing the resulting single- or double-stranded        oligonucleotides to produce a mixture of single-stranded        polynucleotides, optionally separating the shorter or smaller        polynucleotides into pools of polynucleotides having various        lengths and further optionally subjecting said polynucleotides        to a PCR procedure to amplify one or more oligonucleotides        comprised by at least one of said polynucleotide pools;    -   (d) incubating a plurality of said polynucleotides or at least        one pool of said polynucleotides with a polymerase under        conditions which result in annealing of said single-stranded        polynucleotides at regions of identity between the        single-stranded polynucleotides and thus forming of a        mutagenized double-stranded polynucleotide chain;    -   (e) optionally repeating steps (c) and (d);    -   (f) expressing at least one hybrid polypeptide from said        polynucleotide chain, or chains; and    -   (g) screening said at least one hybrid polypeptide for a useful        activity.

In a preferred aspect of the invention, the means for blocking orinterrupting the amplification or synthesis process is by utilization ofuv light, DNA adducts, DNA binding proteins.

In one embodiment of the invention, the DNA adducts, or polynucleotidescomprising the DNA adducts, are removed from the polynucleotides orpolynucleotide pool, such as by a process including heating the solutioncomprising the DNA fragments prior to further processing.

Having thus disclosed exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the disclosures areexemplary only and that various other alternatives, adaptations andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are to be consideredillustrative and thus are not limiting of the remainder of thedisclosure in any way whatsoever.

EXAMPLE 1 Generation of Random Size Polynucleotides Using U.V. InducedPhotoproducts

One microgram samples of template DNA are obtained and treated with U.V.light to cause the formation of dimers, including TT dimers,particularly purine dimers. U.V. exposure is limited so that only a fewphotoproducts are generated per gene on the template DNA sample.Multiple samples are treated with U.V. light for varying periods of timeto obtain template DNA samples with varying numbers of dimers from U.V.exposure.

A random priming kit which utilizes a non-proofreading polymease (forexample, Prime-It II Random Primer Labeling kit by Stratagene CloningSystems) is utilized to generate different size polynucleotides bypriming at random sites on templates which are prepared by U.V. light(as described above) and extending along the templates. The primingprotocols such as described in the Prime-It II Random Primer Labelingkit may be utilized to extend the primers. The dimers formed by U.V.exposure serve as a roadblock for the extension by the non-proofreadingpolymerase. Thus, a pool of random size polynucleotides is present afterextension with the random primers is finished.

EXAMPLE 2 Isolation of Random Size Polynucleotides

Polynucleotides of interest which are generated according to Example 1are are gel isolated on a 1.5% agarose gel. Polynucleotides in the100-300 bp range are cut out of the gel and 3 volumes of 6 M NaI isadded to the gel slice. The mixture is incubated at 50° C. for 10minutes and 10 μl of glass milk (Bio 101) is added. The mixture is spunfor 1 minute and the supernatant is decanted. The pellet is washed with500 μl of Column Wash (Column Wash is 50% ethanol, 10 mM Tris-HCl pH7.5, 100 mM NaCl and 2.5 mM EDTA) and spin for 1 minute, after which thesupernatant is decanted. The washing, spinning and decanting steps arethen repeated. The glass milk pellet is resuspended in 20 μl of H₂O andspun for 1 minute. DNA remains in the aqueous phase.

EXAMPLE 3 Shuffling of Isolated Random Size 100-300bp Polynucleotides

The 100-300 bp polynucleotides obtained in Example 2 are recombined inan annealing mixture (0.2 mM each dNTP, 2.2 mM MgCl₂, 50 mM KCl, 10 mMTris-HCl ph 8.8, 0.1% Triton X-100, 0.3μ; Taq DNA polymerase, 50 μltotal volume) without adding primers. A Robocycler by Stratagene wasused for the annealing step with the following program: 95° C. for 30seconds. 25-50 cycles of [95° C. for 30 seconds, 50-60° C. (preferably58° C.) for 30 seconds, and 72° C. for 30 seconds] and 5 minutes at 72°C. Thus, the 100-300 bp polynucleotides combine to yield double-strandedpolynucleotides having a longer sequence. After separating out thereassembled double-stranded polynucleotides and denaturing them to formsingle stranded polynucleotides, the cycling is optionally againrepeated with some samples utilizing the single strands as template andprimer DNA and other samples utilizing random primers in addition to thesingle strands.

EXAMPLE 4 Screening of Polvpeptides from Shuffled Polynucleotides

The polynucleotides of Example 3 are separated and polypeptides areexpressed therefrom. The original template DNA is utilized as acomparative control by obtaining comparative polypeptides therefrom. Thepolypeptides obtained from the shuffled polynucleotides of Example 3 arescreened for the activity of the polypeptides obtained from the originaltemplate and compared with the activity levels of the control. Theshuffled polynucleotides coding for interesting polypeptides discoveredduring screening are compared further for secondary desirable traits.Some shuffled polynucleotides corresponding to less interesting screenedpolypeptides are subjected to reshuffling.

EXAMPLE 5 Directed Evolution an Enzyme by Saturation Mutagenesis

Site-Saturation Mutagenesis: To accomplish site-saturation mutagenesisevery residue (316) of a dehalogenase enzyme was converted into all 20amino acids by site directed mutagenesis using 32-fold degenerateoligonucleotide primers, as follows:

-   -   1. A culture of the dehalogenase expression construct was grown        and a preparation of the plasmid was made    -   2. Primers were made to randomize each codon—they have the        common structure X₂₀NN(G/T)X₂₀    -   3. A reaction mix of 25 ul was prepared containing ˜50 ng of        plasmid template, 125 ng of each primer, 1X native P fu buffer,        200 uM each dNTP and 2.5 U native Pfu DNA polymerase    -   4. The reaction was cycled in a Robo96 Gradient Cycler as        follows:        -   Initial denaturation at 95° C. for 1 min        -   20 cycles of 95° C. for 45 sec, 53° C. for 1 min and 72° C.            for 11 min        -   Final elongation step of 72° C. for 10 min    -   5. The reaction mix was digested with 10 U of DpnI at 37° C. for        1 hour to digest the methylated template DNA    -   6. Two ul of the reaction mix were used to transform 50 ul of        XL1-Blue MRF′ cells and the entire transformation mix was plated        on a large LB-Amp-Met plate yielding 200-1000 colonies    -   7. Individual colonies were toothpicked into the wells of        96-well microtiter plates containing LB-Amp-IPTG and grown        overnight    -   8. The clones on these plates were assayed the following day

Screening: Approximately 200 clones of mutants for each position weregrown in liquid media (384 well microtiter plates) and screened asfollows:

-   -   1. Overnight cultures in 384-well plates were centrifuged and        the media removed. To each well was added 0.06 mL 1 mM Tris/SO₄        ²⁻ pH 7.8.    -   2. Made 2 assay plates from each parent growth plate consisting        of 0.02 mL cell suspension.    -   3. One assay plate was placed at room temperature and the other        at elevated temperature (initial screen used 55° C.) for a        period of time (initially 30 minutes).    -   4. Afier the prescribed time 0.08 mL room temperature substrate        (TCP saturated 1 mM Tris/SO₄ ²⁻ pH 7.8 with 1.5 mM NaN₃ and 0.1        mM bromothymol blue) was added to each well.    -   5. Measurements at 620 nm were taken at various time points to        generate a progress curve for each well.    -   6. Data were analyzed and the kinetics of the cells heated to        those not heated were compared. Each plate contained 1-2 columns        (24 wells) of unmutated 20F12 controls.    -   7. Wells that appeared to have improved stability were re-grown        and tested under the same conditions.

Following this procedure nine single site mutations appeared to conferincreased thermal stability on the enzyme. Sequence analysis wasperformed to determine of the exact amino acid changes at each positionthat were specifically responsible for the improvement. In sum, theimprovement was conferred at 7 sites by one amino acid change alone, atan eighth site by each of two amino acid changes, and at a ninth site byeach of three amino acid changes. Several mutants were then made eachhaving a plurality of these nine beneficial site mutations incombination; of these two mutants proved superior to all the othermutants, including those with single point mutations.

Literature Cited

-   Alting-Mecs M A and Short J M: Polycos vectors: a system for    packaging filamentous phage and phagemid vectors using lambda phage    packaging extracts. Gene 137:1, 93-100, 1993.-   Arkin A P and Youvan D C: An algorithm for protein engineering:    simulations of recursive ensemble mutagenesis. Proc Natl Acad Sci    USA 89(16):7811-7815, 1992.-   Arnold F H: Protein engineering for unusual environments. Current    Opinion in Biotechnology 4(4):450-455, 1993.-   Ausubel F M, et al. Editors. Current Protocols in Molecular Biology,    Vols. 1 and 2 and supplements. Greene Publishing Assoc., Brooklyn,    N.Y. ©1987.-   Barbas C F 3d, Bain J D, Hoekstra D M, Lemer R A: Semisynthetic    combinatorial antibody libraries: a chemical solution to the    diversity problem. Proc Natl Acad Sci USA 89(10):4457-4461, 1992.-   Bardwell A J, Bardwell L, Johnson D K, Friedberg E C: Yeast DNA    recombination and repair proteins Rad 1 and Rad 10 constitute a    complex in vivo mediated by localized hydrophobic domains. Mol    Microbiol 8(6):1177-1188, 1993.-   Bartel P, Chien C T, Sternglanz R, Fields S: Elimination of false    positives that arise in using the two-hybrid system. Biotechniques    14(6):920-924, 1993.-   Beaudry A A and Joyce G F: Directed evolution of an RNA enzyme.    Science 257(5070):635-641, 1992.-   Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to    Molecular Cloning Techniques. Academic Press, Inc., San Diego,    Calif. ©1987. (Cumulative Subject Index: Volumes 135-139, 141-167    1990, 272 pp., $136.00/ISBN: 0-12-182076-9)-   Biocca S, Pierandrei-Amaldi P, Cattaneo A: Intracellular expression    of anti-p2lras single chain Fv fragments inhibits meiotic maturation    of xenopus oocytes. Biochem Biophys Res Commun 197(2):422-427, 1993.-   Bogerd H P, Fridell R A, Blair W S, Cullen B R: Genetic evidence    that the Tat proteins of human immunodeficiency virus types 1 and 2    can multimerize in the eukaryotic cell nucleus. J Virol    67(8):5030-5034, 1993.-   Breitling F, Dubel S, Seehaus T, Klewinghaus I, Little M: A surface    expression vector for antibody screening. Gene 104(2):147-153, 1991.-   Burton D R, Barbas C F 3d, Persson M A, Koenig S, Chanock R M,    Lerner R A: A large array of human monoclonal antibodies to type 1    human immunodeficiency virus from combinatorial libraries of    asymptomatic seropositive individuals. Proc Natl Acad Sci USA    88(22):10134-10137, 1191.-   Caldwell R C and Joyce G F: Randomization of genes by PCR    mutagenesis. PCR Methods Appl 2(10):28-33, 1992.-   Caton A J and Koprowski H: Influenze virus hemagglutinin-specific    antibodies isolatedf froma combinatorial expression library are    closely related to the immune response of the donor. Proc Natl Acad    Sci USA 87(16):6450-6454, 1990.-   Chakraborty T, Martin J F, Olson E N: Analysis of the    oligomerization of myogenin and E2A products in vivo using a    two-hybrid assay system. J Biol Chem 267(25):17498-501, 1992.-   * Chang et al. J. Immunol. 147: 3610, 1991.-   Chaudhary V K, Batra J K, Gallo M G, Willingham M C, FitzGerald D J,    Pastan I: A rapid method of cloning functional variable-region    antibody genes in Escherichia coli as single-chain immunotoxins.    Proc Natl Acad Sci USA 87(3):1066-1070, 1990.-   Chien C T, Bartel P L, Stemglanz R, Fields S: The two-hybrid system:    a method to identify and clone genes for proteins that interact with    a protein of interest. Proc Natl Acad Sci USA 88(21):9578-9582,    1991.-   Chiswell D J, McCafferty J: Phage antibodies: will new ‘coliclonal’    antibodies replace monoclonal antibodies? Trends Biotechnol    10(3):80-84, 1992.-   Chothia C and Lesk A M: Canonical structures for the hypervariable    regions of immunoglobulins. J Mol Biol 196)4):901-917, 1987.-   Chothia C, Lesk A M, Tramontano A, Levitt M, Smith-Gill S J, Air G,    Sheriff S, Padlan E A, Davies D, Tulip W R, et al.: Conformations of    immunoglobulin hypervariable regions. Nature 342(6252):877-883,    1989.-   Clackson T, Hoogenboom H R, Griffiths A D, Winter G: Making antibody    fragments using phage display libraries. Nature 352(6336):624-628,    1991.-   Dasmahapatra B, DiDomenico B, Dwyer S, Ma J, Sadowski I, Schwartz J:    A genetic system for studying the activity of a proteolytic enzyme.    Proc Natl Acad Sci USA 89(9):4159-4162, 1992.-   Davis L G, Dibner M D, Battey J F. Basic Methods in Molecular    Biology. Elsevier, New York, N.Y. ©1986.-   Delegiave S and Youvan D C. Biotechnology Research 11:1548-1552,    1993.-   DeLong E F, Wu K Y, Prezelin B B, Jovine R V: High abundance of    Archaea in Antarctic marine picoplankton. Nature 371(6499):695-697,    1994.-   Deng S J, MacKenzie C R, Sadowska J, Michniewicz J, Young N M,    Bundle Dr, Narang S A: Selection of antibody single-chain variable    fragments with improved carbohydrate binding by phage display. J    Biol Chem 269(13):9533-9538, 1994.-   Duan L, Bagasra O, Laughlin M A, Oakes J W, Pomerantz R J: Potent    inhibition of human immunodeficiency virus type 1 replication by an    intracellular anti-Rev single-chain antibody. Proc Natl Acad Sci USA    91(11):5075-5079, 1994.-   Durfee T, Becherer K, Chen P L, Yeh S H, Yang Y, Kilburn A E, Lee W    H, Elledge S J: The retinoblastoma protein associates with the    protein phosphatase type 1 catalytic subunit. Genes Dev    7(4):555-569, 1993.-   Ellington A D and Szostak J W: In vitro selection of RNA molecules    that bind specific ligands. Nature 346(6287):818-822, 1990.-   Fields S and Song O: A novel genetic system to detect    protein-protein interactions. Nature 340(6230):245-246, 1989.-   Firek S, Draper J, Owen M R, Gandecha A, Cockburn B, Whitelam G C:    Secretion of a functional single-chain Fv protein in transgenic    tobacco plants and cell suspension cultures. Plant Mol Biol    23(4):861-870, 1993.-   Germino F J, Wang Z X, Weissman S M: Screening for in vivo    protein-protein interactions. Proc Natl Acad Sci USA 90(3):933-937,    1993.-   Gluzman Y: SV40-transformed simian cells support the replication of    early SV40 mutants. Cell 23(1):175-182, 1981.-   Gruber M, Schodin B A, Wilson E R, Kranz D M: Efficient tumor cell    lysis mediated by a bispecific single chain antibody expressed in    Escherichia coli. J Immunol 152(11):5368-5374, 1994.-   Guarente L: Strategies for the identification of interacting    proteins. Proc Natl Acad Sci USA 90(5):1639-1641, 1993.-   Hardy C F, Sussel L, Shore D: A RAP1-interacting protein involved in    transcriptional silencing and telomere length regulation. Genes Dev    6(5):801-814, 1992.-   Hawkins R E and Winter G: Cell selection strategies for making    antibodies from variable gene libraries: trapping the memory pool.    Eur J Immunol 22(3):867-870, 1992.-   Holvoet P, Laroche Y, Lijnen H R, Van Hoef B, Brouwers E, De Cock F,    Lauwereys M, Gansemans Y, Collen D: Biochemical characterization of    single-chain chimeric plasminogen activators consisting of a    single-chain Fv fragment of a fibrin-specific antibody and    single-chain urokinase. Eur J Biochem 210(3 ):945-952, 1992.-   *Honjo, Alt, and THE. Rabbits, eds. Academic Press: San Diego,    Calif., pp. 361-368. 1989.-   Hoogenboom H R, Griffiths A D, Johnson K S, Chiswell D J, Judson P,    Winter G: Multi-subunit proteins on the surface of filamentous    phage: methodologies for displaying antibody (Fab) heavy and light    chains. Nucleic Acids Res 19(15):4133-4137, 1991.-   Huse W D, Sastry L, Iverson S A, Kang A S, Alting-Mees M, Burton D    R, Benkovic S J, Lemer R A: Generation of a large combinatorial    library of the immunoglobulin repertoire in phage lambda. Science    246(4935):1275-1281, 1989.-   Huston J S, Levinson D, Mudgett-Hunter M, Tai M S, Novotney J,    Margolies M N, Ridge R J, Bruccoleri R E, Haber E, Crea R, et al.:    Protein engineering of antibody binding sites: recovery of specific    activity in an anti-digoxin single-chain Fv analogue produced in    Escherichia coli. Proc Natl Acad Sci USA 85(16):5879-5883, 1988.-   Iwabuchi K, Li B, Bartel P, Fields S: Use of the two-hybrid system    to identify the domain of p53 involved in oligomerization. Oncogene    8(6):1693-1696, 1993.-   Jackson A L, Pahl P M, Harrison K, Rosamond J, Sclafani R A: Cell    cycle regulation of the yeast Cdc7 protein kinase by association    with the Dbf4 protein. Mol Cell Biol 13(5):2899-2908, 1993.-   Johnson S and Bird R E: Methods Enzymol 203:88, 1991.-   Kabat et al.: Sequences of Proteins of Immunological Interest, 4th    Ed. U.S. Department of Health and Human Services, Bethesda, Md.    (1987)-   Kang A S, Barbas C F, Janda K D, Benkovic S J, Lerner R A: Linkage    of recognition and replication functions by assembling combinatorial    antibody Fab libraries along phage surfaces. Proc Natl Acad Sci USA    88(10):4363-4366, 1991.-   Kettleborough C A, Ansell K H, Allen R W, Rosell-Vives E, Gussow D    H, Bendig M M: Isolation of tumor cell-specific single-chain Fv from    immunized mice using phage-antibody libraries and the    re-construction of whole antibodies from these antibody fragments.    Eur J Immunol 24(4):952-958, 1994.-   Lalo D, Carles C, Sentenac A, Thuriaux P: Interactions between three    common subunits of yeast RNA polymerases I and III. Proc Natl Acad    Sci USA 90(12):5524-5528, 1993.-   Lefkovits I and Pernis B, Editors. Immunological Methods, Vols. I    and II. Academic Press, New York, N.Y. ©1979.-   Lerner R A, Kang A S, Bain J D, Burton D R, Barbas C F 3d:    Antibodies without immunization. Science 258(5086): 1313-1314, 1992.-   Leung, D. W., et al., Technique, 1:11-15, 1989.-   Li B and Fields S: Identification of mutations in p53 that affect    its binding to SV40 large T antigen by using the yeast two-hybrid    system. FASEB J 7(10):957-963, 1993.-   Lilley G G, Doelzal O, Hillyard C J, Bernard C, Hudson P J:    Recombinant single-chain antibody peptide conjugates expressed in    Escherichia coli for the rapid diagnosis of HIV. J Immunol Methods    171(2):211-226, 1994.-   Lowman H B, Bass S H, Simpson N, Wells J A: Selecting high-affinity    binding proteins by monovalent phage display. Biochemistry    30(45):10832-10838, 1991.-   Luban J, Bossolt K L, Franke E K, Kalpana G V, Goff S P: Human    immunodeficiency virus type 1 Gag protein binds to cyclophilins A    and B. Cell 73(6):1067-1078, 1993.-   Madura et al.: J Biol Chem 268: 12046, 1993.-   Marks J D, Hoogenboom H R, Bonnert T P, McCafferty J, Griffiths A D,    Winter G: By-passing immunization. Human antibodies from V-gene    libraries displayed on phage. J Mol Biol 222(3):581-597, 1991.-   Marks J D, Griffiths Ad, Malmqvist M, Clackson T P, Bye J M, Winter    G: By-passing immunization: building high affinity human antibodies    by chain shuffling. Biotechnology (N Y) 10(7):779-783, 1992.-   Marks J D, Hoogenboom H R, Griffiths A D, Winter G: Molecular    evolution of proteins on filamentous phage. Mimicking the strategy    of the immune system. J Biol Chem 267(23):16007-16010, 1992.-   McCafferty J, Griffiths A D, Winter G, Chiswell D J: Phage    antibodies: filamentous phage displaying antibody variable domains.    Nature 348(6301):552-554, 1990.-   Miller J H. A Short Course in Bacterial Genetics: A Laboratory    Manual and Handbook for Escherichia coli and Related Bacteria. Cold    Spring Harbor Laboratory Press, Plainview, N.Y. ©1992. p. 445-   Milne G T and Weaver D T: Dominant negative alleles of RAD52 reveal    a DNA repair/recombination complex including Rad51 and Rad52. Genes    Dev 7(9):1755-1765, 1993.-   Mullinax R L, Gross E A, Amberg J R, Hay B N, Hogrefe H H, Kubtiz M    M, Greener A, Alting-Mees M, Ardourel D, Short J M, et al.:    Identification of human antibody fragment clones specific for    tetanus toxoid in a bacteriophage lambda immunoexpression library.    Proc natl Acad Sci USA 87(20):8095-9099, 1990.-   Needleman S B and Wunsch C D: A general method applicable to the    search for similarities in the amino acid sequence of two proteins.    J Mol Biol 48(3):443-453. 1970.-   Nicholls P J, Johnson V G, Andrew S M, Hoogenboom H R, Raus J C,    Youle R J: Characterization of single-chain antibody (sFv)-toxin    fusion proteins produced in vitro in rabbit reticulocyte lysate. J    Biol Chem 268(7):5302-5308, 1993.-   Owens R J and Young R J: The genetic engineering of monoclonal    antibodies. J Immunol Methods 168(2):149-165, 1994.-   Pearson W R and Lipman D J: Improved tools for biological sequence    comparison. Proc Natl Acad Sci USA 85(8):2444-2448, 1988.-   Persson M A, Caothien R H, Burton D R: Generation of diverse    high-affinity human monoclonal antibodies by repertoire cloning.    Proc Natl Acad Sci USA 88(6):2432-2436, 1991.-   Queen C, Foster J, Stauber C, Stafford J: Cell-type specific    regulation of a kappa immunoglobulin gene by promoter and enhance    elements. Immunol Rev 89:49-68, 1986.-   Reidhaar-Olson J F and Sauer R T: Combinatorial cassette mutagenesis    as a probe of the informational content of protein sequences.    Science 241(4861):53-57, 1988.-   Riechmann L and Weill M: Phage display and selection of a    site-directed randomized single-chain antibody Fv fragment for its    affinity improvement. Biochemistry 32(34):8848-8855, 1993.-   Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory    Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,    N.Y. ©1989.-   Scopes R K. Protein Purification: Principles and Practice.    Springer-Verlag, New York, N.Y. ©1982.-   Silver S C and Hunt S W 3d: Techniques for cloning cDNAs encoding    interactive transcriptional regulatory proteins. Mol Biol Rep    17(3):155-165, 1993.-   Smith and Waterman: Adv Appl Math 2: 482, 1981.-   Staudinger J, Perry M, Elledge S J, Olson E N: Interactions among    vertebrate helix-loop-helix proteins in yeast using the two-hybrid    system. J Biol Chem 268(7):4608-4611, 1993.-   Stemmer W P, Morris S K, Wilson B S: Selection of an active single    chain Fv antibody from a protein linker library prepared by    enzymatic inverse PCR. Biotechniques 14(2):256-265, 1993.-   Stemmer W P: DNA shuffling by random fragmentation and reassembly:    in vitro recombination for molecular evolution. Proc Natl Acad Sci    USA 91(22):10747-10751. 1994.-   Thiesen H J and Bach C: Target Detection Assay (TDA): a versatile    procedure to determine DNA binding sites as demonstrated on SPI    protein. Nucleic Acids Res 18(11):3203-3209. 1990.-   Tramontano A, Chothia C, Lesk A M: Framework residue 71 is a major    determinant of the position and conformation of the second    hypervariable region in the VH domains of immunoglobulins. J Mol    Biol 215(1):175-182, 1990.-   Tuerk C and Gold L: Systematic evolution of ligands by exponential    enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science    249(4968):505-510, 1990.-   Vojtek A B, Hollenberg S M, Cooper J A: Mammalian Ras interacts    directly with the serine/threonine kinase Raf. Cell 74(1):205-214,    1993.-   Williams and Barclay, in Immunoglobulin Genes, The Immunoglobulin    Gene Superfamily-   Winnacker E L. From Genes to Clones: Introduction to Gene    Technology. VCH Publishers, New York, N.Y. ©1987.-   Winter G and Milstein C: Man-made antibodies. Nature    349(6307):293-299, 1991.-   Yang et al. (1992) Science 257: 680-   U.S. Pat. No. 4,683,195; Filed Feb. 7, 1986, Issued Jul. 28. 1987.    Mullis K B, Erlich H A, Arnheim N, Horn G T, Saiki R K, Scharf S J:    Process for Amplifying, Detecting, and/or Cloning Nucleic Acid    Sequences.-   U.S. Pat. No. 4,683,202; Filed Oct. 25, 1985, Issued Jul. 28, 1987.    Mullis K B: Process for Amplifying Nucleic Acid Sequences.-   U.S. Pat. No. 4,704,362; Filed Nov. 5, 1979, Issued Nov. 3, 1987.    Itakura K, Riggs A D: Recombinant Cloning Vehicle Microbial    Polypeptide Expression.-   IPN WO 92/03918; Filed Aug. 28, 1991, Published Mar. 19, 1992.    Lonberg N, Kay R M: Transgenic Non-human Animals Capable of    Producing Heterologous Antibodies.-   IPN WO 93/12227; Filed Dec 17, 1992, Published Jun. 24, 1993.    Lonberg, N; Kay R M: Transgenic Non-human Animals Capable of    Producing Heterologous Antibodies.-   IPN WO 94/25585; Filed Apr. 25, 1994, Published Nov. 10, 1994.    Lonberg, N, Kay R M: Transgenic Non-human Animals Capable of    Producing Heterologous Antibodies.-   IPN WO 91/17271; Filed May 1, 1990, Published Nov. 14, 1991. Dower W    J, Cwirla S E: Recombinant Library Screening Methods.-   IPN WO 91/18980; Filed May 13, 1991. Published Dec. 12, 1991. Devlin    J J: Compositions and Methods for Indentifying Biologically Active    Molecules.-   IPN WO 91/19818; Filed Jun. 20, 1990. Published Dec. 26, 1991. Dower    W J, Cwirla S E, Barrett R W: Peptide Library and Screening Systems.-   IPN WO 93/08278; Filed Oct. 15, 1992, Published Apr. 29, 1993.    Schatz P J, Cull M G, Miller J F, Stemmer W P: Peptide Library and    Screening Method.-   IPN WO 92/05258; Filed Sept. 17, 1991, Published Apr. 2, 1992.    Fincher G B: Gene Encoding Barley Enzyme.-   IPN WO 92/14843; Filed Feb. 21, 1992, Published Sept. 3, 1992. Toole    J J, Griffin L C, Bock L C, Latham J A, Muenchau D D, Krawczyk S:    Aptamers Specific for Biomolecules and Method of Making.-   IPN WO 88/08453; Filed Apr. 14, 1988, Published Nov. 3, 1988.    Alakhov J B, Baranov, V I, Ovodov S J, Ryabova L A, Spirin A S:    Method of Obtaining Polypeptides in Cell-Free Translation System.-   IPN WO 90/05785; Filed Nov. 15, 1989, Published May 31, 1990.    Schultz P: Method for Site-Specifically Incorporating Unnatural    Amino Acids into Proteins.-   IPN WO 90/07003; Filed Jan. 27, 1989, Published Jun. 28, 1990.    Baranov V I, Morozov I J, Spirin A S: Method for Preparative    Expression of Genes in a Cell-free System of Conjugated    Transcription/translation.-   IPN WO 91/02076; Filed Jun. 14, 1990, Published Feb. 21, 1991.    Baranov V I, Ryabova L A, Yarchuk O B, Spirin A S: Method for    Obtaining Polypeptides in a Cell-free System.-   IPN WO 91/05058; Filed Oct. 5, 1989, Published Apr. 18, 1991.    Kawasaki G: Cell-free Synthesis and Isolation of Novel Genes and    Polypeptides.-   IPN WO 92/02536; Filed Aug. 1, 1991, Published Feb. 20, 1992. Gold    L, Tuerk C: Systematic Polypeptide Evolution by Reverse Translation.-   IPN WO 92/03918; Filed Aug. 28, 1991, Published Mar. 19, 1992.    Lonberg N, Kay R M: Transgenic Non-human Animals Capable of    Producing Heterologous Antibodies.-   IPN WO 93/12227; Filed Dec. 17, 1992, Published Jun. 24, 1993.    Lonberg N, Kay R M: Transgenic Non-human Animals Capable of    Producing Heterologous Antibodies.-   IPN WO 94/25585; Filed Apr. 25, 1994, Published Nov. 10, 1994.    Lonberg N, Kay R M: Transgenic Non-human Animals Capable of    Producing Heterologous Antibodies.

1. (canceled)
 2. A method of providing a population of recombinednucleic acids, the method comprising: (i) providing a population ofoligonucleotides comprising at least one set of codon-variedoligonucleotides, wherein at least one member of the set of codon-variedoligonucleotides is chemically synthesized using at least trinucleotidesequences and wherein two or more members of the population compriseoverlapping oligonucleotides; (ii) hybridizing at least two of theoverlapping oligonucleotides to each other to provide a population ofhybridized overlapping oligonucleotides, which population of hybridizedoverlapping oligonucleotides comprises at least one codon-variedoligonucleotide; and, (iii) elongating members of the population ofhybridized overlapping oligonucleotides, thereby providing thepopulation of recombined nucleic acids.
 3. The method of claim 2,further comprising selecting at least first and second nucleic acids tobe recombined, wherein the set of codon-varied oligonucleotidescomprises a plurality of codon-varied nucleic acids which correspond tothe first and second nucleic acids.
 4. The method of claim 3, whereinthe first and second nucleic acids are homologous.
 5. The method ofclaim 3, wherein the first and second nucleic acids are non-homologous.6. The method of claim 2, wherein the providing step comprisestrinucleotide synthesis comprising: (a) providing a substrate sequencehaving a 5′ terminus and at least one base, the 5′ terminus and at leastone base having protecting groups thereon; (b) removing the 5′protecting group of the substrate sequence to provide a 5′ deprotectedsubstrate sequence; (c) coupling the 5′ deprotected substrate sequencewith a selected trinucleotide phosphoramidite sequence having a 3′terminus, a 5′ terminus, and three base groups, the 3′ terminus, the 5′prime terminus, and the three base groups having protecting groupsthereon, thereby yielding an extended oligonucleotide sequence; and, (d)repeating steps (b) and (c), wherein the extended oligonucleotidesequence yielded by each repeated step (c) becomes the substratesequence of the next repeated step (b) until a desired codon-variedoligonucleotide is obtained.
 7. The method of claim 2, wherein theproviding step comprises trinucleotide synthesis performed in anautomated synthesizer which automatically performs the steps of: (a)providing a substrate sequence having a 5′ terminus and at least onebase, the 5′ terminus and at least one base having protecting groupsthereon; (b) removing the 5′ protecting group of the substrate sequenceto provide a 5′ deprotected substrate sequence; (c) coupling the 5′deprotected substrate sequence with a selected trinucleotidephosphoramidite sequence having a 3′ terminus, a 5′ terminus, and threebase groups, the 3′ terminus, the 5′ prime terminus, and the three basegroups having protecting groups thereon, thereby yielding an extendedoligonucleotide sequence; and, (d) repeating steps (b) and (c), whereinthe extended oligonucleotide sequence yielded by each repeated step (c)becomes the substrate sequence of the next step (b) until a desiredcodon-varied oligonucleotide is obtained.
 8. The method of claim 7, themethod further comprising inputting character string information intothe automatic synthesizer corresponding to the desired codon-variedoligonucleotides to be obtained.
 9. The method of claim 8, wherein thecharacter string information corresponds to two or more nucleic acids tobe recombined.
 10. The method of claim 2, wherein the providing stepcomprises providing a substrate sequence having a 5′ terminus and atleast one base, the 5′ terminus and at least one base having protectinggroups thereon, the substrate sequence further comprising a 3′ end thatis covalently attached to a solid support.
 11. The method of claim 2,wherein the providing step comprises coupling together one or more of:mononucleotides, trinucleotide phosphoramidite sequences, andoligonucleotides.
 12. The method of claim 2, wherein the providing stepcomprises split-pool synthesis comprising: (a) providing substratesequences, each having a 5′ terminus and at least one base, the 5′terminus and at least one base having protecting groups thereon; (b)removing the 5′ protecting groups of the substrate sequences to provide5′ deprotected substrate sequences; (c) coupling the 5′ deprotectedsubstrate sequences with a population of a selected trinucleotidephosphoramidite sequence, each having a 3′ terminus, a 5′ terminus, andthree base groups, the 3′ terminus, the 5′ terminus, and the three basegroups having protecting groups thereon, thereby yielding extendedoligonucleotide sequences; (d) repeating steps (b) and (c), wherein theextended oligonucleotide sequences yielded by each step (c) become thesubstrate sequences of the next step (b), until extended intermediateoligonucleotide sequences are produced; (e) splitting the extendedintermediate oligonucleotide sequences into two or more separate pools;(f) removing the 5′ protecting groups of the extended intermediateoligonucleotide sequences to provide 5′ deprotected extendedintermediate oligonucleotide sequences in the two or more separatepools; (g) coupling the 5′ deprotected extended intermediateoligonucleotide sequences with one or more selected mononucleotides,trinucleotide phosporamidite sequences, or oligonucleotides in the twoor more separate pools, thereby yielding further extended intermediateoligonucleotide sequences; (h) pooling the further extended intermediateoligonucleotide sequences from the two or more separate pools into asingle pool; and, (i) repeating steps (b) through (h), wherein thefurther extended intermediate oligonucleotide sequences in the singlepool of each step (h) become the substrate sequences of the next step(b), until desired codon-varied oligonucleotides are obtained.
 13. Themethod of claim 12, further comprising selecting at least first andsecond nucleic acids to be recombined, wherein the set of codon-variedoligonucleotides comprises a plurality of codon-varied nucleic acidswhich correspond to the first and second nucleic acids wherein the firstand second nucleic acids are non-homologous and are synthesized usingthe split-pool synthesis format.
 14. The method of claim 13, wherein thenon-homologous first and second nucleic acids are less than 90 percentidentical.
 15. The method of claim 14, wherein the split-pool synthesisformat is module-based with a smallest module being a singletrinucleotide in length and a larger module being at least 15nucleotides in length.
 16. The method of claim 2, wherein the providingstep comprises split-pool synthesis performed in an automatedsynthesizer which automatically performs the steps of: (a) providingsubstrate sequences having a 5′ terminus and at least one base, the 5′terminus and at least one base having protecting groups thereon; (b)removing the 5′ protecting groups of the substrate sequences to provide5′ deprotected substrate sequences; (c) coupling the 5′ deprotectedsubstrate sequences with a population of a selected trinucleotidephosphoramidite sequence having a 3′ terminus, a 5′ terminus, and threebase groups, the 3′ terminus, the 5′ terminus, and the three base groupshaving protecting groups thereon, thereby yielding extendedoligonucleotide sequences; (d) repeating steps (b) and (c), wherein theextended oligonucleotide sequences yielded by each step (c) become thesubstrate sequences of the next step (b), until extended intermediateoligonucleotide sequences are provided; (e) splitting the extendedintermediate oligonucleotide sequences into two or more separate pools;(f) removing the 5′ protecting groups of the extended intermediateoligonucleotide sequences to provide 5′ deprotected extendedintermediate oligonucleotide sequences in the two or more separatepools; (g) coupling the 5′ deprotected extended intermediateoligonucleotide sequences with one or more selected mononucleotides,trinucleotide phosporamidite sequences, or oligonucleotides in the twoor more separate pools, thereby yielding further extended intermediateoligonucleotide sequences; (h) pooling the further extended intermediateoligonucleotide sequences from the two or more separate pools into asingle pool; and, (i) repeating steps (b) through (h), wherein thefurther extended intermediate oligonucleotide sequences in the singlepool of each step (h) become the substrate sequences of the next step(b), until desired codon-varied oligonucleotides are obtained.
 17. Themethod of claim 16, further comprising selecting at least first andsecond nucleic acids to be recombined, wherein the set of codon-variedoligonucleotides comprises a plurality of codon-varied nucleic acidswhich correspond to the first and second nucleic acids wherein the firstand second nucleic acids are non-homologous and are synthesized usingthe split-pool synthesis format performed in an automated synthesizer.18. The method of claim 17, wherein the non-homologous first and secondnucleic acids are less than 90 percent identical.
 19. The method ofclaim 18, wherein the split-pool synthesis format performed in anautomated synthesizer is module-based with a smallest module being asingle trinucleotide in length and a larger module being at least 15nucleotides in length.
 20. The method of claim 16, the method furthercomprising inputting character string information into the automaticsynthesizer corresponding to the desired codon-varied oligonucleotidesto be obtained.
 21. The method of claim 20, wherein the character stringinformation corresponds to two or more nucleic acids to be recombined.22. The method of claim 16, wherein the providing step further comprisesproviding a substrate sequence having a 3′ terminus covalently attachedto a solid support.
 23. The method of claim 2, wherein the hybridizingstep occurs in vitro.
 24. The method of claim 2, wherein the hybridizingstep occurs in vivo.
 25. The method of claim 2, wherein (iii) compriseselongating the one or more members of the population of hybridizedoverlapping codon-varied oligonucleotides with a polymerase.
 26. Themethod of claim 25, wherein the polymerase is a thermostable polymerase.27. The method of claim 2, the method further comprising: denaturing thepopulation of recombined nucleic acids to provide a set of denaturedrecombined nucleic acids; re-hybridizing at least one member of the setof denatured recombined nucleic acids to at least one other member ofthe set of denatured recombined nucleic acids to provide a population ofrehybridized recombined nucleic acids; elongating one or more members ofthe population of rehybridized recombined nucleic acids to provide apopulation of further recombined nucleic acids; and, selecting at leastone member of the population of further recombined nucleic acids for atleast one desired trait or property.
 28. The method of claim 2, themethod further comprising the steps of: denaturing the population ofrecombined nucleic acids to provide a set of denatured recombinednucleic acids; re-hybridizing at least one member of the set ofdenatured recombined nucleic acids to at least one other member of theset of denatured recombined nucleic acids to provide a population ofrehybridized recombined nucleic acids; elongating one or more members ofthe population of re-hybridized recombined nucleic acids to provide apopulation of further recombined nucleic acids; and, repeating thedenaturing, re-hybridizing and elongating steps at least once.
 29. Themethod of claim 28, further comprising selecting at least one member ofthe population of further recombined nucleic acids for at least onedesired trait or property.
 30. The method of claim 28, wherein aplurality of members of the population of recombined nucleic acids areselected for a desired trait or property to provide first round selectednucleic acids, the method further comprising: hybridizing at least onemember of a second set of overlapping codon-varied oligonucleotides toat least one other member of the second set of overlapping codon-variedoligonucleotides to provide a second population of hybridizedoverlapping codon-varied oligonucleotides, which second set ofoverlapping codon-varied oligonucleotides is derived from the firstround selected nucleic acids; and, elongating one or more members of thesecond population of hybridized overlapping codon-variedoligonucleotides to provide a second population of further recombinednucleic acids.
 31. The method of claim 30, further comprising sequencingthe first round selected nucleic acids, wherein the second set ofoverlapping codon-varied oligonucleotides is derived from the firstround selected nucleic acids by aligning sequences of the first roundselected nucleic acids to one another to identify regions of similarityand regions of diversity in the first round selected nucleic acids, andsynthesizing the second set of overlapping codon-varied oligonucleotidesto comprise a plurality of oligonucleotides, each of which comprisesubsequences corresponding to at least one region of diversity.
 32. Themethod of claim 30, wherein the first round selected nucleic acidsencode polypeptides of about 50 amino acids or less.
 33. The method ofclaim 30, wherein the second set of overlapping codon-variedoligonucleotides comprises a plurality of oligonucleotide member typeswhich comprise consensus region subsequences derived from a plurality ofthe first round selected nucleic acids.
 34. The method of claim 2,further comprising selecting at least one member of the population ofrecombined nucleic acids for at least one desired trait or property. 35.The method of claim 2, wherein the set of overlapping codon-variedoligonucleotides comprises a plurality of oligonucleotide member typeswhich comprise consensus region subsequences derived from a plurality ofhomologous target nucleic acids.
 36. The method of claim 2, wherein theset of overlapping codon-varied oligonucleotides comprises at least 3oligonucleotide member types.
 37. The method of claim 36, wherein theset of overlapping codon-varied oligonucleotides comprises at least 5oligonucleotide member types.
 38. The method of claim 37, wherein theset of overlapping codon-varied oligonucleotides comprises at least 10oligonucleotide member types.
 39. The method of claim 2, wherein the setof overlapping codon-varied oligonucleotides comprises a plurality ofhomologous oligonucleotide member types, wherein the homologousoligonucleotide member types are present in equimolar amounts.
 40. Themethod of claim 2, wherein the set of overlapping codon-variedoligonucleotides comprises a plurality of homologous oligonucleotidemember types, wherein the homologous oligonucleotide member types arepresent in non-equimolar amounts.